Polypeptides for promoting cell attachment

ABSTRACT

Novel polypeptides derived from human fibronectin are described which bind to integrin receptors expressed by cells. The receptor binding site of human fibronectin begins at amino acid residue 1394 and ends at residue 1400 of fibronectin. The polypeptides facilitate attachment of cells to substrates either alone or in conjunction with RGD-containing peptides. Vectors, fusion proteins and antibodies are also described. Methods for promoting cell attachment and for inhibiting cell adhesion are also described.

This invention was made with the support of the United StatesGovernment, and the United States Government has certain rights in theinvention pursuant to National Institutes of Health Grant HL 28235.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a continuation-in-part of copendingapplication Ser. No. 07/725,600, filed Jul. 3, 1991, now abandoned whichis a continuation-in-part of application Ser. No. 07/620,668, filed Dec.3, 1990, now abandoned, which applications are hereby incorporated byreference.

DESCRIPTION

1. Technical Field

The present invention relates to methods and compositions for promotingcell attachment to substrates. The invention particularly relates to theuse of newly identified binding sites of fibronectin for binding tointegrin receptors on cells.

2. Background

Regulation of cell adhesive events has broad biomedical implications.For instance, inhibition of cell adhesion may be of benefit in thetreatment of thrombotic disorders through inhibition of plateletaggregation, of inflammatory disorders through inhibition of leukocyteadhesion and transmigration, and in malignant disease through inhibitionof tumor cell lodgement and metastasis. Conversely, promotion of celladhesion is, in some cases, desirable. For example, in the seeding ofendothelial cells onto vascular grafts, in the stability of medicalprostheses, and in promotion of wound healing. Adhesive events arewidely recognized to involve interactions of extracellular receptors,i.e., integrin receptors, with substances surrounding the cell, e.g.,fibronectin. Integrins are a functionally and structurally related groupof receptors that interact with a wide variety of ligands includingextracellular matrix glycoproteins, complement and other cells.Integrins participate in cell-matrix and cell-cell adhesion in manyphysiologically important processes including embryological development,hemostasis, thrombosis, wound healing, immune and nonimmune defensemechanisms and oncogenic transformation. See Hynes, Cell, 48:549-554(1987). Several integrins that participate in dynamic cell adhesion binda tripeptide, arginine-glycine-aspartic acid (RGD), present in theirligand. See Ruoslahti et al., Science, 238:491-497 (1987).

Fibronectin is an adhesive glycoprotein found in plasma and on cellsurfaces and extracellular matrices. By binding other macromolecules aswell as cells, fibronectin promotes anchorage of cells to substrata.Hynes, in Cell Biology of the Extracellular Matrix, Hay ed., PlenumPress, pages 295-334 (1982); Hynes et al., J. Cell Biol., 95:369-77(1982). Also, fibronectin is known to accumulate at sites of injury andinflammation in vivo Pettersson et al., Clin. Immunol. Immunopath,11:425-436 (1978); Grinnel et al., J. Invest. Derm., 76:181-189 (1981);Repesh et al., J. Histochem. Cytochem., 30(4):399-408 (1985); Carsons etal., Arth. Rheum, 24(10):1261-67 (1981)! and is produced by cells inblood vessel walls at these sites. Clark et al., J. Exp. Med.,156:646-51 (1982); Clark et al., J. Immunol., 126(2):787-93 (1981);Clark et al., J. Invest. Derm., 79:269-76 (1982); Clark et al., J. ClinInvest., 74:1011-16 (1984).

Fibronectin is composed of subunits of variable primary structureaverage relative molecular mass of 250 kilodaltons (kDa)!. The subunitsare disulfide-linked to form dimers or multimers derived from a pool ofsimilar but nonidentical polypeptides. Hynes, in Cell Biology of theExtracellular Matrix, Hay ed., Plenum Press, pages 295-334 (1982); Hyneset al., Cell Biol., 95:369-77 (1982); Schwarzbauer et al., Proc. Natl..Acad. Sci. USA, 82:1424-28; Kornblihtt et al., EMBO J., 4(7): 1755-59(1985). Thus, the term "fibronectin" refers to several species ofglycoprotein, some of which are more fully characterized than others.

Two major fibronectin (Fn) classes are plasma fibronectin and cellularfibronectin. Plasma fibronectin (pFn) is secreted by hepatocytes,whereas cellular fibronectin (cFn) is secreted by a variety of culturedcells including endothelial cells and fibroblasts. Jaffe et al., J. ExpMed., 147:1779-91 (1978); Birdwell et al., Biochem. Biophys. Res.Commun., 97(2):574-8 (1980). Despite extensive physical and immunologicsimilarities, the two classes of fibronectin differ in electrophoreticbehavior, solubility, and biologic activities. Tamkun et al., J. Biol.Chem., 258 (7):4641-47 (1983); Yamada et al., J. Cell Biol., 80:492-98(1979); Yamada et al., Biochemistry, 16 (25):2552-59, (1977).

Primary structural differences between plasma and cellular fibronectinshave been found by peptide mapping Hayashi et al., J. Biol. Chem.,256(21):11,292-11,300 (1981)!, CDNA cloning Kornblihtt et al., EMBO J.,4:1755-1759 (1985)!, and immunologic techniques Atherton et al., Cell,25:133-41 (1981)!. From these data, it has been determined that theprimary structure of fibronectin monomer contains three different typesof internal repeats known as homology Types I, II and III, havinglengths of about 40, 60 and 90 amino acids residues, respectivelyKornblihtt et al., EMBO J., 4:1755-1759 (1985)!. All of the variousdistinct Fn moieties are produced by a single gene, with differences inprimary structure resulting from alternative splicing of the primarymRNA transcript in at least three regions. Kornblihtt et al., EMBO J.,4(7):1755-59 (1985); Schwarzbauer et al., Proc. Natl. Acad. Sci. USA,82:1424-28 (1985); Gutman et al., Proc. Natl. Acad. Sci. USA, 84:7179-82(1987); Schwarzbauer et al., EMBO J., 6(9):2573-80 (1987).

A site containing the Arg-Gly-Asp (RGD) sequence in the 10th Type IIIrepeat of Fn is known to be involved in cell adhesive events. Peptidescontaining this sequence inhibit certain cell adhesive events, oralternatively, can be used to promote cell adhesion. See, e.g., U.S.Pat. Nos. 4,589,881; 4,661,111; 4,517,686; 4,683,291; 4,578,079;4,614,517; and 4,792,525.

Recently, site-directed mutagenesis studies of fibronectin haveimplicated non-RGD sequences as participating in cell adhesionphenomena. Obara, M. et al. Cell, 53:649-57 (1988)!. The proposed secondbinding site was not defined by this study; however, activity loss dataindicated that a second site was involved in adhesion, probably in asynergistic fashion with the RGD sequence. This result helps to explainwhy other RGD-containing proteins do not bind integrins as well asfibronectin.

In view of the importance of promoting cell adhesion or, conversely, forinhibiting adhesion, non-RGD containing polypeptides suitable for thesepurposes are desired. In the event such polypeptides are found tocomplement RGD amino acid residue sequences in cell binding processes,compositions including both RGD sequences and adhesive non-RGD compoundsare also desired.

BRIEF SUMMARY OF THE INVENTION

The present invention is for polypeptides that bind to integrinreceptors, particularly GPIIb-IIIa, which polypeptides comprise abinding region for the integrin that is independent of the well-knownRGD sequence of fibronectin (Fn). The new binding site is located atleast fifty amino acid residues upstream (toward the N-terminus) of theRGD sequence of human Fn. The amino acid residue sequence of human Fn isdescribed in Kornblihtt, et al., EMBO J., 4:1755 (1985), whichdescription is incorporated herein by reference. Selected regions ofhuman Fn are depicted in FIGS. 1-3 and include the sequence offibronectin described by Kornblihtt et al.

In one embodiment, the present invention contemplates a polypeptidehaving a length of no more than about 100 amino acid residues. Thepeptide binds GPIIb-IIIa and includes an amino acid residue sequencerepresented by the formula: --DRX₁ PHX₂ R--, where X₁ and X₂ are anyamino acid residue (SEQ ID NO 1).

Preferably, the polypeptide includes an amino acid residue sequencerepresented by the formula: --DRX₁ PHX₂ RU--, wherein X₁ is V or A, X₂is S or A, and U is a sequence of amino acids represented by formulaselected from the group consisting of: --X₃ X₄ X₅ X₆ --, --X₃ SIT--,--NX₄ IT--, --NSX₅ T--, and --NSIX₆ --, respectively, SEQ ID NO 2through 6, wherein X₃ is N or A, X₄ is S or A, X₅ is I or A, and X₆ is Tor A.

A preferred embodiment contemplates a polypeptide shown in SEQ ID NOs2-6 which have the respective amino acid residue sequences of DRX₁ PHX₂RX₃ X₄ X₅ X₆, DRX₁ PHX₂ RX₃ SIT, DRX₁ PHX₂ RNX₄ IT, DRX₁ PHX₂ RNSX₅ Tand DRX₁ PHI₂ RNSIX₆.

In a preferred embodiment of the invention the instant polypeptides willhave an amino acid residue sequence represented by the formula B--X--Zwhere X is the amino acid residue sequence shown in FIG. 1 (residues1351-1456 of human Fn) (SEQ ID NO 7), B is an NH₂ group or N-terminalsequence of amino acids, and Z is a COOH group or C-terminal sequence ofamino acids no more than 150 residues in length.

In another embodiment of the invention, the instant polypeptides willhave an amino acid residue sequence represented by formula J--U--X--Zwhere X and Z are as described above, J is an NH₂ group or N-terminalamino acid residue sequence, and U is the amino acid residue sequence ofhuman Fn from residues 1255-1350 depicted in FIG. 2 which corresponds toamino acid residues positions 1-96 in SEQ ID NO 8.

The instant polypeptides can bind to GPIIb-IIIa independently or inconcert with an RGD-containing peptide. When binding is complementarywith an RGD sequence the RGD sequence may be incorporated in the sameprotein as the instant polypeptides or it may be provided in a distinctpeptide.

Also contemplated within the invention are methods for attaching cells,e.g., endothelial cells, to a substrate in which the method comprisescontacting cells expressing GPIIb-IIIa with the substrate comprising theinstant polypeptides affixed to a solid matrix and maintaining thecontact for a predetermined time sufficient for the GPIIb-IIIa to bindthe polypeptides. Use of the polypeptide-substrate is contemplated inskin grafting and prosthesis.

Also contemplated are vectors for expressing the instant polypeptidesand fusion proteins of the polypeptides. One embodiment of the inventionallows ready purification of the polypeptides by generating maltosebinding protein (MBP) fusion products of the polypeptides.

A still further embodiment contemplates antibody compositions thatimmunoreact with the instant polypeptides which can competitivelyinhibit Fn or fibrinogen (Fg) binding to GPIIb-IIIa. The instantpolypeptides may also be used to inhibit Fn or Fg binding to GPIIb-IIIa.

Thus, the present invention affords novel polypeptides and relatedcompositions and methods which promote cell attachment and/or inhibitcell adhesion.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the 1351-1456 amino acid residue sequence of human Fn(residues 1-106 of SEQ ID NO 7).

FIG. 2 depicts the 1255-1456 amino acid residue sequence of human Fn(residues 1-202 of SEQ ID NO 8).

FIGS. 3A and 3B depict the 1255-1456 amino acid residue sequence ofhuman Fn and a corresponding double-stranded DNA sequence coding for theamino acid residue sequence according to the principles of the presentinvention.

The nucleic acid coding strand of the human Fn sequence and the encodedamino acid residue sequence are listed in the Sequence Listing as SEQ IDNO 9, with Fn residues 1255-1456 shown as residues 1-202 of SEQ ID NO 9.The nucleic acid non-coding strand is listed in the 5' to 3' directionas SEQ ID NO 10.

FIG. 4A is a graph that shows the effect on binding of Fn to immobilizedGPIIb-IIIa by increasing concentrations (μg/ml) of monoclonal antibodies(Nabs) raised to Fn according to the principles of the presentinvention. The data is expressed as a percent of binding as described inthe Examples.

FIGS. 4B, 4C, and 4D are graphs that show cross competition studies oflabelled Mabs for sites on Fn in the presence of unlabelled Mabs.Competition is measured as an amount of lab (cpm×10³) binding toimmobilized Fn in the presence of the indicated Nabs: Nab 8 (3), Nab 16(16), Mab 11.10 (11), Nab 15 (15), or no Nab (No).

FIG. 5 shows MBP vectors pIH821 and pPR734 for synthesizing GPIIIa-MBPfusion proteins, and relevant cloning sites.

FIG. 6 shows the effect of Fn binding to immobilized GPIIb-IIIa in thepresence of added fibronectin (Fn; open squares), maltose-bindingprotein (MBP; closed circles) alone, MBP-(Fn residue sequence 1255-1456)fusion protein (MBP/III 8+9; open circles), and control BSA alone(closed squares).

FIG. 7 shows the effect of various concentrations (μg/ml) of MBP fusionprotein (MBP/III 8+9) versus MBP on Fn binding and fibrinogen (Fg)binding to immobilized GPIIb-IIIa. Closed circles indicates inhibitionof FG binding in the presence of the fusion protein, and open circlesindicates control inhibition in the presence of MBP. Open squaresindicates inhibition of Fn binding in the presence of the fusionprotein, and closed squares indicates control inhibition in the presenceof MBP.

FIG. 8 illustrates the competitive inhibition of Fn binding to purifiedGPIIb-IIIa by the Fn-derived polypeptide D-11-T having the amino acidresidue sequence DRVPHSRNSIT (SEQ ID NO 11) as shown by the line withclosed squares. Also shown is the inhibition of Fn binding to GPIIb-IIIaby the Fn-derived polypeptide RGDS (SEQ ID NO 12) indicated by the linewith open squares. The competition assays were performed in microtiterwells of a 96-well plate as described in Example 9b(1). Briefly,microtiter wells coated with purified GPIIb-IIIa receptor weremaintained with 10 nM biotinylated Fn for 2 hours at room temperature inthe presence of varying concentrations of D-11-T and RGDS. The amount ofbound biotinylated fibronectin was determined using avidin bound tobiotinylated horseradish peroxidase H as a disclosing reagent. Theamount of fibronectin bound is reported as absorbance at 490 nm.Fibronectin in the absence of competitive inhibitor polypeptides yieldedan absorbance 490 or 2.70. RGDS and D-11-T maximally inhibited thebinding of Fn to GPIIb-IIIa at a concentration of 20 μM and 200 μM,respectively.

FIG. 9 illustrates the effects of alanine substitutions in the D-11-Tpolypeptide on the inhibitory activity of preventing the binding of Fnto GPIIb-IIIa. The quantity of 10 nM biotinylated Fn bound to microtiterwells coated with purified GPIIb-IIIa in the presence of 250 μMpolypeptide was determined as described in FIG. 8 and in Example 9b(2).The percent inhibitory effect of D-11-T polypeptide, the scrambledcontrol peptide VHPDRNTISRS (SEQ ID NO 13), and the polypeptides with analanine substituted at the amino acid residue below each bar in the bargraph are reported. Results presented represent the average plus/minusthe standard deviation of three determinations. The results arediscussed in Example 9b(2).

FIG. 10A and 10B illustrate in two panels, 10A and 10B, the inhibitoryactivity of D-11-T polypeptide on the binding of both Fn and fibrinogen(Fg) to GPIIb-IIIa but not to the vitronectin receptor, αvβ3. Thebinding of 5 nM biotinylated Fg is shown in panel 10A and the binding of10 nM biotinylated Fn is shown in panel 10B. The competition assays areperformed as described in FIG. 8 and in Example 9b(3). Inhibition curvesfor assays performed in microtiter wells coated with GPIIb-IIIa areshown in both panels with lines indicated by open and closed squares.Inhibition curves for assays performed in microtiter wells coated withαvβ3 are shown in both panels with lines indicated by open and closedcircles. Open squares and circles show the inhibitory activity of D-11-Tpolypeptide on GPIIb-IIIa and αvβ3, respectively. Closed squares andcircles show the inhibitory activity of the scrambled controlpolypeptide VHPDRNTISRS (SEQ ID NO 13) on GPIIb-IIIa and αvβ3,respectively. Results presented represent triplicate determinationsplus/minus the standard deviation as percent binding of Fg or Fnrelative to the amount bound in the absence of inhibitor. The resultsare discussed in Example 9b(3).

DETAILED DESCRIPTION OF THE INVENTION

A. Definitions

Amino Acid Residue: An amino acid formed upon chemical digestion(hydrolysis) of a polypeptide at its peptide linkages. The amino acidresidues described herein are preferably in the "L" isomeric form.However, residues in the "D" isomeric form can be substituted for anyL-amino acid residue, as long as the desired functional property isretained by the polypeptide. NH₂ refers to the free amino group presentat the amino terminus of a polypeptide. COOH refers to the free carboxygroup present at the carboxy terminus of a polypeptide. In keeping withstandard polypeptide nomenclature (described in J. Biol. Chem.,243:3552-59 (1969) and adopted at 37 C.F.R. §1.822(b)(2)), abbreviationsfor amino acid residues are shown in the following Table ofCorrespondence:

    ______________________________________                                        TABLE OF CORRESPONDENCE                                                       SYMBOL                                                                        1-Letter      3-Letter    AMINO ACID                                          ______________________________________                                        Y             Tyr         tyrosine                                            G             Gly         glycine                                             F             Phe         phenylalanine                                       M             Met         methionine                                          A             Ala         alanine                                             S             Ser         serine                                              I             Ile         isoleucine                                          L             Leu         leucine                                             T             Thr         threonine                                           V             Val         valine                                              P             Pro         proline                                             K             Lys         lysine                                              H             His         histidine                                           Q             Gln         glutamine                                           E             Glu         glutamic acid                                       Z             Glx         Glu and/or Gln                                      W             Trp         tryptophan                                          R             Arg         arginine                                            D             Asp         aspartic acid                                       N             Asn         asparagine                                          B             Asx         Asn and/or Asp                                      C             Cys         cysteine                                            X             Xaa         Unknown or other                                    ______________________________________                                    

It should be noted that all amino acid residue sequences representedherein by formulae have a left to right orientation in the conventionaldirection of amino-terminus to carboxy-terminus. In addition, the phrase"amino acid residue" is broadly defined to include the amino acidslisted in the Table of Correspondence and modified and unusual aminoacids, such as those listed in 37 C.F.R. §1.822(b)(4), and incorporatedherein by reference. Furthermore, it should be noted that a dash at thebeginning or end of an amino acid residue sequence indicates a peptidebond to a further sequence of one or more amino acid residues or to anamino-terminal group such as NH₂ or to a carboxy-terminal group such asCOOH.

Antibody: a polypeptide which chemically binds to a haptenic group,i.e., ligand. Antibodies, as used herein, are immunoglobulin moleculesand immunologically active fragments of immunoglobulin molecules. Suchportions known in the art as Fab, Fab'; F(ab')₂ and F_(v) are included.Typically, antibodies bind ligands that range in size from about 6 toabout 34 Å with association constants in the range of about 10⁴ to 10¹⁰M⁻¹ and as high as 10¹² M⁻¹. Antibodies may be polyclonal or monoclonal(MoAb). Antibodies can bind a wide range of ligands, including smallmolecules such as steroids and prostaglandins, biopolymers such asnucleic acids, proteins and polysaccharides, and synthetic polymers suchas polypropylene. An "antibody combining site" is that structuralportion of an antibody molecule comprised of a heavy and light chainvariable and hypervariable regions that specifically binds (immunoreactswith) antigen. The term "immunoreact" in its various forms is usedherein to refer to binding between an antigenic determinant-containingmolecule and a molecule containing an antibody combining site such as awhole antibody molecule or a portion thereof. An "antigenic determinant"is the actual structural portion of the antigen that is immunologicallybound by an antibody combining site. The term is also usedinterchangeably with "epitope".

Ligand: a molecule that contains a structural portion that is bound byspecific interaction with a particular receptor molecule.

Oligonucleotide or Polynucleotide: a polymer of single or doublestranded nucleotides. As used herein "oligonucleotide" and itsgrammatical equivalents will include the full range of nucleic acids. Anoligonucleotide will typically refer to a nucleic acid moleculecomprised of a linear strand of two or more deoxyribonucleotides and/orribonucleotides. The exact size will depend on many factors, which inturn depends on the ultimate conditions of use, as is well known in theart.

Polypeptide or Peptide: a linear series of at least two amino acidresidues in which adjacent residues are connected by peptide bondsbetween the alpha-amino group of one residue and the alpha-carboxy groupof an adjacent residue.

Protein: refers to a linear series of more than 50 amino acid residuesin which adjacent residues are connected via peptide linkages.

Receptor: a biologically active proteinaceous molecule that specificallybinds to (or with) other molecules (ligands). Receptors can beglycosylated.

Vector: a DNA molecule capable of autonomous replication in a cell andto which a DNA segment, e.g., gene or polynucleotide, can be operativelylinked so as to bring about replication of the attached segment. Vectorscapable of directing the expression of DNA segments (genes) encoding oneor more proteins are referred to herein as "expression vectors". Vectorsalso allow cloning of cDNA (complementary DNA) from mRNAs produced usingreverse transcriptase.

B. Polypeptides

The polypeptides of the present invention include an amino acid residuesequence corresponding to a region of fibronectin (Fn) and are referredto as fibronectin-derived polypeptides or Fn polypeptides.

The Fn used in this invention was isolated from fresh human citratedplasma by affinity chromatography on gelatin-sepharose according to themethods described by Plow et al., J. Biol. Chem., 256:9477-9482 (1981)and in U.S. Pat. No. 4,589,981. The isolated Fn yielded a single band onSDS-PAGE under nonreducing conditions and a closely spaced doublet of215,000 to 230,000 molecular weight under reducing conditions.Hereinafter, Fn refers to intact isolated Fn as described above and inExample 9b(1).

Typically, the subject polypeptides corresponding to a region of Fn willnot contain an RGD sequence, thereby presenting potential binding sitesfor ligands that have a three-dimensional structure different from theRGD sequence of Fn. It is preferred that the entire sequence of thepolypeptide represent a portion of Fn.

In one embodiment, a polypeptide of this invention has a length of nomore than 100 amino acid residues and is characterized by the presenceof a sequence represented by the formula:

    --DRX.sub.1 PHX.sub.2 R--,

(SEQ ID NO 1) where X₁ and X₂ are any amino acid residue, preferably anamino acid residue selected from the Table of Correspondence.Preferably, X₁ and X₂ are independently selected from the residues ofGly, Ala, Val, Ser and Thr. Preferably, X₁ and X₂ are independentlyselected from the residues of Lys, Arg and His. Preferably, X₁ and X₂are independently selected from the residues of Asp, Gln, Glu, Asn, Cysand Met. Preferably, X₁ and X₂ are independently selected from theresidues of Pro, Tyr and Trp.

A preferred polypeptide in this embodiment has an amino acid residuesequence represented by the formula DRVPHSR (SEQ ID NO 16).

A preferred polypeptide further includes a carboxy-terminal amino acidsequence U, i.e., has the formula --DRX₁ PHX₂ RU--, where X₁ is Val orAla and X₂ is Ser or Ala. U is one of the amino acid residue sequences--X₃ X₄ X₅ X₆ --, X₃ SIT--, --NX₄ IT--, --NSX₅ T-- and --NSIX₆ --,respectively, SEQ ID NOs 2 through 6, wherein X₃ is Asn or Ala, X₄ isSer or Ala, X₅ is Ile or Ala and X₆ is Thr or Ala.

A preferred embodiment contemplates a polypeptide shown in SEQ ID NOs2-6 which have the respective amino acid residue sequences of DRX₁ PHX₂RX₃ X₄ X₅ X₆, DRX₁ PHX₂ RX₃ SIT, DRX₁ PHX₂ RNX₄ IT, DRX₁ PHX₂ RNSX₅ Tand DRX₁ PHI₂ RNSIX₆. Preferably, the amino- and carboxy-terminal dashesof the formula --DRX₁ PHX₂ RU-- are amino- and carboxy-terminal groups,respectively, with the preferred amino-terminal group being NH₂, and thepreferred carboxy-terminal group being COOH.

Preferably, the polypeptides will include an amino acid residue sequencethat corresponds to the sequence of residues 1351-1456 of Fn (FIG. 1)(SEQ ID NO 7). Also, the polypeptides usually, but not always, willinclude amino acid residues at either or both the amino or carboxy endof the 1351-1456 sequence of Fn, e.g., the 1255-1456 sequence shown inFIG. 2 (SEQ ID NO 8). Additionally it is preferred that the COOH end ofthe present polypeptides extend no more than about 150 residues past thecarboxy end of the amino acid residue sequence depicted in FIGS. 1 or 3(SEQ ID NO 7 and 9, respectively).

A preferred polypeptide in this embodiment has an amino acid residuesequence represented by a formula selected from the group consisting of:

    DRVPHSRNSIT,

    DRAPHSRNSIT,

    DRVPHARNSIT,

    DRVPHSRASIT,

    DRVPHSRNAIT,

    DRVPHSRNSAT, and

    DRVPHSRNSIA,

the SEQ ID NO of which are 11, 17, 18, 19, 20, 21 and 22, respectively.

In one embodiment of the invention the polypeptide can have amaltose-binding protein (MBP) covalently bonded to the N-terminus of theselected Fn sequence. The MBP region of the fusion protein stronglybinds to immobilized amylose (starch) which facilitates purification ofthe desired protein from containments, such as non-MBP containingproteins. The MBP fragment may be directly bonded to the selected Fnfragment or intervening amino acid residues may be provided between theMBP and polypeptide regions.

In one embodiment, the instant polypeptides are not glycosylated, i.e.,they are produced directly by peptide synthesis techniques or areproduced in a procaryotic cell transformed with a recombinant DNA of thepresent invention. Eucaryotically produced peptide molecules aretypically glycosylated.

It should be understood that a subject polypeptide need not be identicalto the amino acid residue sequence of Fn, so long as the subjectpolypeptides are able to compete for binding sites of GPIIb-IIIa.

A subject polypeptide includes any analog, fragment or chemicalderivative of a polypeptide whose amino acid residue sequence is shownherein so long as the polypeptide is able to compete for binding sitesof GPIIb-IIIa. Therefore, a present polypeptide can be subject tovarious changes, insertions, deletions and substitutions, eitherconservative or non-conservative, where such changes provide for certainadvantages in its use.

In this regard, a polypeptide of this invention corresponds to, ratherthan is identical to, the sequence of Fn where one or more changes aremade and it retains the ability to compete for binding sites in one ormore of the assays as defined herein.

The term "analog" includes any polypeptide having an amino acid residuesequence substantially identical to a sequence specifically shown hereinin which one or more residues have been conservatively substituted witha functionally similar residue and which displays the ability to inhibitbinding as described herein. Examples of conservative substitutionsinclude the substitution of one non-polar (hydrophobic) residue such asisoleucine, valine, leucine or methionine for another, the substitutionof one polar (hydrophilic) residue for another such as between arginineand lysine, between glutamine and asparagine, between glycine andserine, the substitution of one basic residue such as lysine, arginineor histidine for another, or the substitution of one acidic residue,such as aspartic acid or glutamic acid for another.

The phrase "conservative substitution" also includes the use of achemically derivatized residue in place of a non-derivatized residueprovided that such polypeptide displays the requisite activity.

"Chemical derivative" refers to a subject polypeptide having one or moreresidues chemically derivatized by reaction of a functional side group.Such derivatized molecules include for example, those molecules in whichfree amino groups have been derivatized to form amine hydrochlorides,p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonylgroups, chloroacetyl groups or formyl groups. Free carboxyl groups maybe derivatized to form salts, methyl and ethyl esters or other types ofesters or hydrazides. Free hydroxyl groups may be derivatized to formO-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine maybe derivatized to form N-im-benzylhistidine. Also included as chemicalderivatives are those peptides which contain one or more naturallyoccurring amino acid derivatives of the twenty standard amino acids. Forexamples: 4-hydroxyproline may be substituted for proline;5-hydroxylysine may be substituted for lysine; 3-methylhistidine may besubstituted for histidine; homoserine may be substituted for serine; andornithine may be substituted for lysine.

Particularly preferred modifications are those modifications designed toincrease the stability of the polypeptide in solution, and thereforeserve to prolong half life of the polypeptides in solutions,particularly biological fluids such as blood, plasma or serum. Exemplarymodifications are those that block susceptibility to proteolyticactivity in the blood. Thus a polypeptide can have a stabilizing groupat one or both termini. Typical stabilizing groups include amido,acetyl, benzyl, phenyl, tosyl, alkoxycarbonyl, alkyl carbonyl,benzyloxycarbonyl and the like end group modifications. Additionalmodifications include using a "L" amino acid in place of a "D" aminoacid at the termini, cyclization of the polypeptide, and amide ratherthan amino or carboxy termini to inhibit exopeptidase activity.

Polypeptides of the present invention also include any polypeptidehaving one or more additions and/or deletions or residues relative tothe sequence of a polypeptide whose sequence is shown herein, so long asthe requisite activity is maintained.

The term "fragment" refers to any subject polypeptide having an aminoacid residue sequence shorter than that of a polypeptide whose aminoacid residue sequence is shown herein.

When a polypeptide of the present invention has a sequence that is notidentical to the sequence of Fn, it is typically because one or moreconservative or non-conservative substitutions have been made, usuallyno more than about 30 number percent, and preferably no more than 10number percent of the amino acid residues are substituted.

"Substantially homologous" means that a particular subject sequence ormolecule, for example, a mutant sequence, varies from a referencesequence by one or more substitutions, deletions, or additions, the neteffect of which does not result in an adverse functional dissimilaritybetween reference and subject sequences. For purposes of the presentinvention, amino acid sequences having greater than 90 percentsimilarity, equivalent biological activity, and equivalent expressioncharacteristics are considered substantially homologous and are includedwithin the scope of a polypeptide of this invention.

Amino acid sequences having greater than 40 percent similarity areconsidered substantially similar. For purposes of determining homologyor similarity, truncation or internal deletions of the referencesequence should be disregarded, as should subsequent modifications ofthe molecule, e.g., glycosylation. Sequences having lesser degrees ofhomology and comparable bioactivity are considered equivalents.

Additional residues may also be added at either terminus of anpolypeptide of this invention for the purpose of providing a "linker" bywhich the polypeptides of this invention can be conveniently affixed toa label or solid matrix, or carrier. Preferably, the linker residues donot form epitopes which are cross reactive with Fn, i.e., are notsufficiently similar in structure to a Fn polypeptide as to producecross-reacting antibodies.

Labels, solid matrices and carriers that can be used with thepolypeptides of this invention are described hereinbelow.

Amino acid residue linkers are usually at least one residue and can be40 or more residues, more often 1 to 10 residues, but do not formepitopes cross-reactive with a Fn polypeptide of this invention. Typicalamino acid residues used for linking are tyrosine, cysteine, lysine,glutamic and aspartic acid, or the like. In addition, a subjectpolypeptide can differ, unless otherwise specified, from the naturalsequence of the corresponding protease by the sequence being modified byterminal-NH₂ acylation, e.g., acetylation, or thioglycolic acidamidation, by terminal-carboxylamidation, e.g., with ammonia,methylamine, and the like terminal modifications.

When coupled to a carrier to form what is known in the art as acarrier-hapten conjugate, a polypeptide of the present invention iscapable of inducing antibodies that immunoreact with Fn. In view of thewell established principle of immunologic cross-reactivity, the presentinvention therefore contemplates antigenically related variants of apolypeptide of this invention. An "antigenically related variant" is asubject polypeptide that is capable of inducing antibody molecules thatimmunoreact with a polypeptide of this invention and immunoreact withFn.

Any peptide of the present invention may be used in the form of apharmaceutically acceptable salt. Suitable acids which are capable offorming salts with the peptides of the present invention includeinorganic acids such as hydrochloric acid, hydrobromic acid, perchloricacid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric aceticacid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalicacid, malonic acid, succinic acid, maleic acid, fumaric acid,anthranilic acid, cinnamic acid, naphthalene sulfonic acid, sulfanilicacid or the like.

Suitable bases capable of forming salts with the peptides of the presentinvention include inorganic bases such as sodium hydroxide, ammoniumhydroxide, potassium hydroxide and the like; and organic bases such asmono-, di- and tri-alkyl and aryl amines (e.g. triethylamine,diisopropyl amine, methyl amine, dimethyl amine and the like) andoptionally substituted ethanolamines (e.g. ethanolamine, diethanolamineand the like).

A polypeptide of the present invention also referred to herein as asubject polypeptide, can be synthesized by any of the techniques thatare known to those skilled in the polypeptide art, including recombinantDNA techniques. Synthetic chemistry techniques, such as a solid-phaseMerrifield-type synthesis, are preferred for reasons of purity,antigenic specificity, freedom from undesired side products, ease ofproduction and the like. An excellent summary of the many techniquesavailable can be found in J. M. Steward and J. D. Young, "Solid PhasePeptide Synthesis", W. H. Freeman Co., San Francisco, 1969; M.Bodanszky, et al., "Peptide Synthesis", John Wiley & Sons, SecondEdition, 1976 and J. Meienhofer, "Hormonal Proteins and Peptides", Vol.2, p. 46, Academic Press (New York), 1983 for solid phase peptidesynthesis, and E. Schroder and K. Kubke, "The Peptides", Vol. 1,Academic Press (New York), 1965 for classical solution synthesis, eachof which is incorporated herein by reference. Appropriate protectivegroups usable in such synthesis are described in the above texts and inJ. F. W. McOmie, "Protective Groups in Organic Chemistry", Plenum Press,New York, 1973, which is incorporated herein by reference.

In general, the solid-phase synthesis methods contemplated comprise thesequential addition of one or more amino acid residues or suitablyprotected amino acid residues to a growing peptide chain. Normally,either the amino or carboxyl group of the first amino acid residue isprotected by a suitable, selectively removable protecting group. Adifferent, selectively removable protecting group is utilized for aminoacids containing a reactive side group such as lysine.

Using a solid phase synthesis as exemplary, the protected or derivatizedamino acid is attached to an inert solid support through its unprotectedcarboxyl or amino group. The protecting group of the amino or carboxylgroup is then selectively removed and the next amino acid in thesequence having the complimentary (amino or carboxyl) group suitablyprotected is admixed and reacted under conditions suitable for formingthe amide linkage with the residue already attached to the solidsupport. The protecting group of the amino or carboxyl group is thenremoved from this newly added amino acid residue, and the next aminoacid (suitably protected) is then added, and so forth. After all thedesired amino acids have been linked in the proper sequence, anyremaining terminal and side group protecting groups (and solid support)are removed sequentially or concurrently, to afford the finalpolypeptide.

Alternatively, the instant polypeptides may be synthesized byrecombinant DNA techniques. A number of different nucleotide sequencesmay code for a particular amino acid residue sequence due to theredundancy of the genetic code. Such nucleotide sequences are consideredfunctionally equivalent since they can result in the production of thesame amino acid residue sequence in all organisms. Occasionally, amethylated variant of a purine or pyrimidine may be incorporated into agiven nucleotide sequence. However, such methylations do not affect thecoding relationship in any way.

C. DNA Segments

Contemplated within the present invention are deoxyribonucleic acid(DNA) molecules that define a gene coding for, i.e., capable ofexpressing, a subject polypeptide or a subject chimeric polypeptide. DNAmolecules that encode the subject polypeptides can easily be synthesizedby chemical techniques, for example, the phosphotriester method ofMatteucci et al., J. Am. Chem. Soc., 103:3185 (1981). Of course, bychemically synthesizing the coding sequence, any desired modificationscan be made simply by substituting the appropriate bases for thoseencoding the native amino acid residue sequence.

A DNA molecule that includes a DNA sequence encoding a subjectpolypeptide can be prepared by operatively linking (ligating)appropriate restriction fragments from each of the above depositedplasmids using well known methods. The DNA molecules of the presentinvention produced in this manner typically have cohesive termini, i.e.,"overhanging" single-stranded portions that extend beyond thedouble-stranded portion of the molecule. The presence of cohesivetermini on the DNA molecules of the present invention is preferred.

Also contemplated by the present invention are ribonucleic acid (RNA)equivalents of the above described DNA molecules.

Preferred DNA segments will encode polypeptides that include an aminoacid residue sequence corresponding to that shown in FIG. 1 (residues1351-1456 of Fn) (SEQ ID NO 7).

D. Vectors

The present invention further contemplates a recombinant DNA moleculecomprising a vector operatively linked, for replication and/orexpression, to a subject DNA molecule, i.e., a DNA molecule defining agene coding for a subject polypeptide or a subject chimeric polypeptide.

The choice of vector to which a DNA segment of the present invention isoperatively linked depends directly, as is well known in the art, on thefunctional properties desired, e.g., protein expression, and the hostcell to be transformed, these being limitations inherent in the art ofconstructing recombinant DNA molecules. However, a vector contemplatedby the present invention is at least capable of directing thereplication, and preferably also expression, of the subject chimericpolypeptide gene included in DNA segments to which it is operativelylinked.

In preferred embodiments, a vector contemplated by the present inventionincludes a procaryotic replicon, i.e., a DNA sequence having the abilityto direct autonomous replication and maintenance of the recombinant DNAmolecule extrachromosomally in a procaryotic host cell, such as abacterial host cell, transformed therewith. Such replicons are wellknown in the art. In addition, those embodiments that include aprocaryotic replicon also include a gene whose expression confers drugresistance to a bacterial host transformed therewith. Typical bacterialdrug resistance genes are those that confer resistance to ampicillin ortetracycline.

Those vectors that include a procaryotic replicon can also include aprocaryotic promoter capable of directing the expression (transcriptionand translation) of the subject chimeric polypeptide gene in a bacterialhost cell, such as E. coli, transformed therewith. A promoter is anexpression control element formed by a DNA sequence that permits bindingof RNA polymerase and transcription to occur. Promoter sequencescompatible with bacterial hosts, such as a tac promoter, are typicallyprovided in plasmid vectors containing convenient restriction sites forinsertion of a DNA segment of the present invention. Typical of suchvector plasmids are pUC8, pUC9, pBR322 and pBR329 available from BioradLaboratories, (Richmond, Calif.) and pPL and pKK223 available fromPharmacia (Piscataway, N.J.).

Expression vectors compatible with eucaryotic cells, preferably thosecompatible with vertebrate cells, can also be used to form therecombinant DNA molecules of the present invention. Eucaryotic cellexpression vectors are well known in the art and are available fromseveral commercial sources. Typically, such vectors are providedcontaining convenient restriction sites for insertion of the desired DNAsegment. Typical of such vectors are pSVL and pKSV-10 (Pharmacia),pBPV-1pML2d (International Biotechnologies, Inc.), and pTDT1 (ATCC,#31255).

In preferred embodiments, the eucaryotic cell expression vectors used toconstruct the recombinant DNA molecules of the present invention includea selection marker that is effective in an eucaryotic cell, preferably adrug resistance selection marker. A preferred drug resistance marker isthe gene whose expression results in neomycin resistance, i.e., theneomycin phosphotransferase (neo) gene. Southern et al., J. Mol. Appl.Genet., 1:327-341 (1982).

The use of retroviral expression vectors to form the rDNA of the presentinvention is also contemplated. As used herein, the term "retroviralexpression vector" refers to a DNA molecule that includes a promotersequence derived from the long terminal repeat (LTR) region of aretrovirus genome.

In preferred embodiments, the expression vector is typically aretroviral expression vector that is preferably replication-incompetentin eucaryotic cells. The construction and use of retroviral vectors hasbeen described by Sorge, et al., Mol. Cell. Biol., 4:1730-37 (1984).

A variety of methods have been developed to operatively link DNA tovectors via complementary cohesive termini. For instance, complementaryhomopolymer tracts can be added to the DNA segment to be inserted and tothe vector DNA. The vector and DNA segment are then joined by hydrogenbonding between the complementary homopolymeric tails to formrecombinant DNA molecules.

Synthetic linkers containing one or more restriction sites provide analternative method of joining the DNA segment to vectors. The DNAsegment, generated by endonuclease restriction digestion as describedearlier, is treated with bacteriophage T4 DNA polymerase of E. coli DNApolymerase I, enzymes that remove protruding, 3', single-strandedtermini with their 3'-5' exonucleolytic activities and fill in recessed3' ends with their polymerizing activities. The combination of theseactivities therefore generates blunt-ended DNA segments. The blunt-endedsegments are then incubated with a large molar excess of linkermolecules in the presence of an enzyme that is able to catalyze theligation of blunt-ended DNA molecules, such as bacteriophage T4 DNAligase. Thus, the products of the reaction are DNA segments carryingpolymeric linker sequences at their ends. These DNA segments are thencleaved with the appropriate restriction enzyme and ligated to anexpression vector that has been cleaved with an enzyme that producestermini compatible with those of the DNA segment.

Synthetic linkers containing a variety of restriction endonuclease sitesare commercially available from a number of sources includingInternational Biotechnologies, Inc. (New Haven, Conn.).

Also contemplated by the present invention are RNA equivalents of theabove described recombinant DNA molecules.

Preferred vectors will include a DNA segment as shown in FIG. 3, whichencodes a polypeptide of the present invention. Exemplary vectorsinclude pIH821 and pPR734 (FIG. 5), for preparing MBP fusion proteinsaccording to the principles of this invention.

E. Transformation of Hosts

The present invention also relates to host cells transformed with arecombinant DNA (rDNA) molecule of the present invention preferably anrDNA capable of expressing a subject chimeric polypeptide. The host cellcan be either procaryotic or eucaryotic. Bacterial cells are preferredprocaryotic host cells and typically are a strain of E. coli such as,for example, the E. coli strain DH5 available from Bethesda ResearchLaboratories, Inc., Bethesda, Md. Preferred eucaryotic host cellsinclude yeast and mammalian cells, preferably vertebrate cells such asthose from a mouse, rat, monkey or human fibroblastic cell line.Preferred eucaryotic host cells include Chinese hamster ovary (CHO)cells available from the ATCC as CCL61 and NIH Swiss mouse embryo cellsNIH/3T3 available from the ATCC as CRL 1658. Transformation ofappropriate cell hosts with a recombinant DNA molecule of the presentinvention is accomplished by well known methods that typically depend onthe type of vector used. With regard to transformation of procaryotichost cells, see, for example, Cohen et al., Proc. Natl. Acad. Sci. USA,69:2110 (1972); and Maniatis et al., Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982).With regard to transformation of vertebrate cells with retroviralvectors containing rDNAs. See, for example, Sorge et al., Mol. Cell.Biol., 4:1730-37 (1984); Graham et al., Virol., 52:456 (1973); andWigler et al., Proc. Natl. Acad. Sci. USA, 76:1373-76 (1979).

Successfully transformed cells, i.e., cells that contain a recombinantDNA molecule of the present invention, can be identified by well knowntechniques. For example, cells resulting from the introduction of anrDNA of the present invention can be cloned to produce monoclonalcolonies. Cells from those colonies can be harvested, lysed and theirDNA content examined for the presence of the rDNA using a method such asthat described by Southern, J. Mol. Biol., 98-503 (1975) or Berent etal., Biotech., 3:208 (1985).

In addition to directly assaying for the presence of rDNA, successfultransformation can be confirmed by well known immunological methods whenthe rDNA is capable of directing the expression of a subject chimericpolypeptide. Samples of cells suspected of being transformed areharvested and assayed for the presence of polypeptide antigenicity usinganti-Fn antibodies.

In addition to the transformed host cells themselves, the presentinvention also contemplates a culture of those cells, preferably amonoclonal (clonally homogeneous) culture, or a culture derived from amonoclonal culture, in a nutrient medium.

F. Expression and Purification

Nutrient media useful for culturing transformed host cells are wellknown in the art and can be obtained from several commercial sources. Inembodiments wherein the host cell is mammalian, a "serum-free" medium ispreferably used.

Methods for recovering an expressed protein from a culture are wellknown in the art and include fractionation of the protein-containingportion of the culture using well known biochemical techniques. Forinstance, the methods of gel filtration, gel chromatography,ultrafiltration, electrophoresis, ion exchange, affinity chromatographyand the like, such as are known for protein fractionations, can be usedto isolate the expressed proteins found in the culture. In addition,immunochemical methods, such as immunoaffinity, immunoadsorption and thelike can be performed using well known methods. A preferred purificationmethod employs immobilized Mabs to Fn.

G. Therapeutic Methods and Compositions

A subject polypeptide can be used in a composition for promoting theattachment (adhesion) of cells to a substrate. Based on the ability of asubject polypeptide to bind with an integrin on the cells, the subjectpolypeptide provides a means for binding to the receptor, and thereforecan be used to promote cell attachment activity when the polypeptide isimmobilized onto a substrate. A composition containing a subjectpolypeptide is used to treat a substrate and thereby to immobilize thepolypeptide contained in the composition onto the substrate.

The substrate can be any solid-matrix having a surface on which celladhesion promoting activity is desired and includes containers for cellculture, medical devices, prosthetic devices, synthetic resin fibers,blood vessels or vascular grafts, percutaneous devices, artificialorgans, and the like. The surface can additionally be comprised ofglass, a synthetic resin, nitrocellulose, polyester, agarose, collagenor a long chain polysaccharide.

Immobilization of polypeptides onto substrate can be accomplished by avariety of means and depends, inter alia, on the substrate and themechanism of immobilization desired. Methods for polypeptideimmobilization or coupling to the substrate are well known in the artand typically involve covalent linkages between a thiol or amino groupon the polypeptide to a reactive group present on the substrate. Forexamples of polypeptide immobilization methods see Aurameas et al.,Scand J. Immunol., Vol. 8 Suppl. 7:7-23 (1978); U.S. Pat. Nos.4,493,795, 4,578,079 and 4,671,950; Klipstein et al., J. Infect. Dis.,147:318-326 (1983) and Liu et al., Biochem., 80:690 (1979). For examplesof the use of cell adhesion promoting polypeptides see U.S. Pat. No.4,578,079.

Also contemplated are prosthetic and medical devices that make use ofthe substrata to attach cells to the surface in vivo or to promotegrowth of cells on a particular surface prior to grafting. For example,endothelial cell growth can be induced on prosthetic blood vessels orvascular grafts, such as those woven or knitted from polyester fibers.Such devices can be useful for wound closure and healing followingaccidents or surgery. In such cases it may be useful to couple thepolypeptides to other biological molecules, such as collagen,glycosaminoglycans, etc. The coupling can be facilitated by chemicalcrosslinking, e.g., by disulfide bridges. Surfaces of prosthetic devicescan also be coated with the instant polypeptides, particularly when thedevices are intended for use temporarily in the body, e.g., forinsertion into blood vessels or into the peritoneal cavity.

The subject polypeptides can be provided within a wide variety ofcompositions. Thus, the polypeptide compositions can comprise one ormore polypeptides as well as a suitable application medium, such as agel, salve, lotion, colloid or powder. The composition is applied to thesubstrate using conventional means and the cells desired to be attachedare applied using techniques well-known to the skilled practitioner.

A related embodiment contemplates modulating the adhesion in vivo ofcells presenting an integrin receptor recognized by the polypeptide. Forinstance, a subject polypeptide can be used in a pharmaceuticallyacceptable composition that, when administered to a human subject in aneffective amount, is capable of competitively inhibiting the aggregationof platelets. That inhibition is believed to result in a decreased rateof thrombus formation. Thus, in vivo administration of a subjectpolypeptide can be used to modulate any physiological response initiatedby adhesion such as coagulation and some inflammatory responses.

In another embodiment, the normal cellular adhesion functions of a cellbearing an integrin on its surface can be inhibited or modulated byintravenous administration of an effective amount of a pharmaceuticallyacceptable composition comprising a polyclonal or monoclonal antibody ofthis invention that immunoreacts with polypeptide.

Insofar as polyclonal or monoclonal antibodies can be usedtherapeutically to modulate cell adhesion-mediated events, the presentinvention also contemplates the use of a subject polypeptide toneutralize the modulating effect of therapeutically administeredantibodies, e.g., as an antidote for the anti-polypeptide antibody. Thechoice of polypeptide to be administered as an antidote depends upon theantibody to be neutralized, and requires that the administeredpolypeptide have the capacity to immunoreact with the administeredantibody.

The polypeptide- or antibody molecule-containing compositionsadministered take the form of solutions or suspensions, however,polypeptides can also take the form of tablets, pills, capsules,sustained release formulations or powders.

A therapeutic composition typically contains an amount of at least 0.1weight percent of active ingredient, i.e., a polypeptide or antibody ofthis invention, per weight of total therapeutic composition. A weightpercent is a ratio by weight of active ingredient to total composition.Thus, for example, 0.1 weight percent is 0.1 grams of polypeptide per100 grams of total composition.

Stated differently, a therapeutic composition typically contains about0.1 micromolar (μM) to about 1.0 molar (M) of polypeptide as activeingredient, preferably about 1.0 to about 100 millimolar (mM), whereasthe antibody molecule-containing compositions typically contain about0.1 to about 20 milligram of antibody as active ingredient permilliliter of therapeutic composition, and preferably about 1 mg/ml toabout 10 mg/ml.

The preparation of a therapeutic composition that contains polypeptidesor antibody molecules as active ingredients is well understood in theart. Typically, such compositions are prepared as injectables, either asliquid solutions or suspensions, however, solid forms suitable forsolution in, or suspension in, liquid prior to injection can also beprepared. The preparation can also be emulsified. The active therapeuticingredient is often mixed with excipients which are pharmaceuticallyacceptable and compatible with the active ingredient as are well known.Suitable excipients are, for example, water, saline, dextrose, glycerol,ethanol, or the like and combinations thereof. In addition, if desired,the composition can contain minor amounts of auxiliary substances suchas wetting or emulsifying agents, pH buffering agents which enhance theeffectiveness of the active ingredient.

A polypeptide or antibody can be formulated into the therapeuticcomposition as neutralized pharmaceutically acceptable salt forms.Pharmaceutically acceptable salts include the acid addition salts(formed with the free amino groups of the polypeptide or antibodymolecule) that are formed with inorganic acids such as, for example,hydrochloric or phosphoric acids, or such organic acids as acetic,tartaric, mandelic, and the like. Salts formed with the free carboxylgroups can also be derived from inorganic bases such as, for example,sodium, potassium, ammonium, calcium, or ferric hydroxides, and suchorganic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol,histidine, procaine, and the like.

The therapeutic polypeptide- or antibody-containing compositions areconventionally administered intravenously, as by injection of a unitdose, for example. The term "unit dose" when used in reference to atherapeutic composition of the present invention refers to physicallydiscrete units suitable as unitary dosages for humans, each unitcontaining a predetermined quantity of active material calculated toproduce the desired therapeutic effect in association with the requireddiluent, i.e., carrier, or vehicle.

The compositions are administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount. The quantity tobe administered depends on the subject to be treated, capacity of thesubject to utilize the active ingredient, and degree of inhibition ofreceptor-ligand binding desired. Precise amounts of active ingredientrequired to be administered depend on the judgment of the practitionerand are peculiar to each individual. However, suitable dosage ranges areof the order of one to several milligrams of active ingredient perindividual per day and depend on the route of administration. Suitableregimes for initial administration and booster shots are also variable,but are typified by an initial administration followed by repeated dosesat one or more hour intervals by a subsequent injection or otheradministration. Alternatively, continuous intravenous infusionsufficient to maintain therapeutically effective concentrations in theblood are contemplated. For a subject polypeptide, therapeuticallyeffective blood concentrations are in the range of about 1.0 mM to about10 mM, preferably about 50 mM to about 1.0 mM. Whenever, the subjectpolypeptides are used for promoting attachment of cells, e.g., to bindendothelial cells, such compositions will typically have a higherconcentration than those taken internally.

A therapeutically effective amount of a polypeptide of this invention istypically an amount of polypeptide such that when administered in aphysiologically tolerable composition is sufficient to achieve a plasmaconcentration of from about 1 micromolar (uM) to about 100 millimolar(mM), and preferably from about 10 uM to about 100 uM.

A therapeutically effective amount of an antibody of this invention istypically an amount of antibody such that when administered in aphysiologically tolerable composition is sufficient to achieve a plasmaconcentration of from about 0.1 microgram (ug) per milliliter (ml) toabout 100 ug/ml, and preferably from about 1 ug/ml to about 5 ug/ml.

H. Antibodies and Monoclonal Antibodies

A reagent of the instant invention is a molecule that specifically bindsan epitope of fibronectin (Fn) defined by a polypeptide of thisinvention. As used herein, the term "specific binding" and itsgrammatical equivalents refers to a non-random binding reaction betweena receptor and a ligand molecule. Illustrative of a specifically-boundreceptor-ligand complex as contemplated herein is that between plateletreceptor GPIIb-IIIa and ligand Fn at the platelet surface. Otherreagents known to specifically bind Fn include polyclonal and monoclonalantibodies raised against Fn and antigenic fragments thereof. Thus,suitable antibodies for producing an antibody of this invention includenative antibodies for Fn and antibodies raised against antigenicdeterminants of Fn such as those defined by the polypeptides of thisinvention.

The different functional regions of fibronectin can be effectivelyprobed by the use of antibodies to the fibronectin molecule. Thus,antibodies raised to fibronectin can be screened for their ability tobind to (immunoreact) various polypeptide fragments of fibronectin. Whenthe antibody composition studied binds with a given fragment, thefragment is identified as presenting an epitope for the antibody. Inthis way, the different regions of the protein molecule which arerecognized by a given antibody molecule are identified.

The instant Fn polypeptides additionally bind preferentially to theirreceptors. As used herein, a reagent molecule of the instant inventionis regarded as "preferentially binding" a target species in the assaywhen the reagent more strongly associates with the target molecule thanwith other species present in the assay. Thus, the reaction of reagentmolecule with target generally will have a greater association constantthan the reaction of reagent with any other species present in theassay. Typically, a reagent herein will "preferentially" bind its targetspecies when the binding affinity of the reagent for target is 2-3 foldgreater, and preferably at least 10 times greater, than thecorresponding affinity of the reagent for another species. Thus, amonoclonal antibody (Mab) to Fn will preferably have a ten fold greateraffinity to Fn than to control peptides. Conversely, Fn preferably bindsthe instant Mabs more than ten times greater than control Mabs.

An antibody composition of the present invention is characterized ascontaining antibody molecules that immunoreact with Fn and fragmentsthereof, and immunoreact with a polypeptide of this invention, but donot preferentially bind (immunoreact) with other species of polypeptide,such as a polypeptide derived from the sequence of Fn and containing theEight Type III repeat sequence. An exemplary polypeptide has thesequence of Fn between residues 1235 and 1325. In a preferredembodiment, the antibody molecules immunoreact with a non-RGD-containingamino acid sequence of Fn located at least 50 residues upstream (towardthe N-terminus) of the RGD sequence of Fn. Preferably, the antibodycompositions will immunoreact with polypeptides that include the aminoacid residue sequence shown in FIG. 1 (SEQ ID NO 7).

A preferred antibody as contemplated herein is typically produced byimmunizing a mammal with an inoculum containing Fn, or polypeptidefragments thereof, from a preselected host animal, thereby inducing inthe mammal antibody molecules having the appropriate immunospecificityfor the target antigen. The antibody molecules are then collected fromthe mammal and screened to the extent desired by well known techniquessuch as, for example, by immunoaffinity for immobilized Fn. Furthermore,an antibody of this invention can be screened for its ability to inhibitbinding of a Fn ligand to a GPIIb-IIIa receptor using standardcompetitive inhibition assays, such as are described in the Examples.The antibody composition so produced can be used inter alia, in thediagnostic methods and systems of the present invention to detect theantigen in a bodily fluid sample.

An antibody of this invention therefor immunoreacts with the GPIIb-IIIabinding site on Fn as defined herein and thereby inhibits for binding toits native receptor GPIIb-IIIa, providing its utility as reagent forinhibiting Fn binding to GPIIb-IIIa.

Thus, a preferred polyclonal antibody is characterized as having theability to immunoreact with a Fn subunit and thereby inhibit thecapacity of the Fn to specifically bind to its receptor, preferably theFn receptor GPIIb-IIIa. Thus, a subject polyclonal antibody is useful toinhibit, and thereby modulate, either in vivo or in vitro, the adhesionof cells which contain integrin receptors that bind to Fn.

A polyclonal antibody of the present invention is typically produced byimmunizing a mammal with an inoculum of the present invention,preferably an inoculum containing a peptide incorporating an amino acidresidue sequence located at least 50 amino acids upstream of RGD. Theantibody molecules are then collected from the mammal and isolated tothe extent desired by well known techniques such as, for example, byimmunoaffinity chromatography using the immunizing polypeptide in thesolid phase. The polyclonal antibody so produced can be used in, interalia, the diagnostic methods and systems of the present invention todiscriminate between Fn and other proteins or between Fn fragmentscontaining epitopes of the antibodies and other fragments, etc. Theantibodies can also be used in therapeutic methods for the purpose ofmodulating cell adhesion, such as inhibiting platelet adhesion.

Monoclonal antibodies (Mabs) to Fn are also contemplated by the presentinvention. The phrase "monoclonal antibody composition" in its variousgrammatical forms refers to a population of antibody molecules thatcontain only one species of antibody combining site capable ofimmunoreacting with a particular antigen. The instant Mab compositionthus typically displays a single binding affinity for any antigen withwhich it immunoreacts. A monoclonal antibody composition may thereforecontain an antibody molecule having a plurality of antibody combiningsites, each immunospecific for a different antigen, e.g., a bispecificmonoclonal antibody.

Mabs of the present invention are typically composed of antibodiesproduced by clones of a single cell, called a hybridoma, that secretes(produces) but one kind of antibody molecule. The hybridoma cell isformed by fusing an antibody-producing cell and a myeloma or otherself-perpetuating cell line. Such antibodies were first described byKohler and Milstein, Nature, 256:495-497 (1975), which description isincorporated by reference.

A monoclonal antibody can also be produced by methods well known tothose skilled in the art of producing chimeric antibodies. Those methodsinclude isolating, manipulating, and expressing the nucleic acid thatcodes for all or part of an immunoglobulin variable region includingboth the portion of the variable region comprising the variable regionof immunoglobulin light chain and the portion of the variable regioncomprising the variable region of immunoglobulin heavy chain. Methodsfor isolating, manipulating, and expressing the variable region codingnucleic acid in procaryotic and eucaryotic hosts are disclosed inRobinson et al., PCT Publication No. WO 89/0099; Winter et al., EuropeanPatent Publication No. 0239400; Reading, U.S. Pat. No. 4,714,681;Cabilly et al., European Patent Publication No. 0125023; Sorge et al.,Mol. Cell Biol., 4:1730-1737 (1984); Beher et al., Science,240:1041-1043 (1988); Skerra et al., Science, 240:1030-1041 (1988); andOrlandi et al., Proc. Natl. Acad. Sci. USA, 86: 3833-3837 (1989).Typically the nucleic acid codes for all or part of an immunoglobulinvariable region that binds a preselected antigen (ligand). Sources ofsuch nucleic acid are well known to one skilled in the art and, forexample, may be obtained from a hybridoma producing a monoclonalantibody that binds the preselected antigen, or the preselected antigenmay be used to screen an expression library coding for a plurality ofimmunoglobulin variable regions, thus isolating the nucleic acid.

Preferred monoclonal antibodies immunoreact with free Fn or polypeptidefragments thereof. The antibodies may also immunoreact with Fn fragmentsimmobilized on substrates or bound to a ligand, such as GPIIb-IIIa, aslong as the Mab epitope(s) is not occluded.

The present invention also contemplates a method of forming a monoclonalantibody molecule that immunoreacts with a region of Fn.

(a) Immunizing an animal with a Fn polypeptide of this invention in theform of animmunogen. Preferably, the immunogen is a homologous sample ofpolypeptides as described herein. However, the antigen may also belinked to a carrier protein such as keyhole limpet hemocyanin,particularly when the antigen is small. The immunization is typicallyaccomplished by administering the sample to an immunologically competentmammal in an immunologically effective amount, i.e., an amountsufficient to produce an immune response. Preferably, the mammal is arodent such as a rabbit, rat or mouse. The mammal is then maintained fora time period sufficient for the mammal to produce cells secretingantibody molecules that immunoreact with the receptor.

(b) A suspension of antibody-producing cells removed from the immunizedmammal is then prepared. This is typically accomplished by removing thespleen of the mammal and mechanically separating the individual spleencells in a physiologically tolerable medium using methods well known inthe art.

(c) The suspended antibody-producing cells are treated with atransforming agent capable of producing a transformed ("immortalized")cell line. Transforming agents and their use to produce immortalizedcell lines are well known in the art and include DNA viruses such asEpstein Bar Virus (EBV), Simian Virus 40 (SV40), Polyoma Virus and thelike, RNA viruses such as Moloney Murine Leukemia Virus (Mo-MuLV), RousSarcoma Virus and the like, myeloma cells such as P3X63-Ag8.653,Sp2/O-Ag14 and the like.

In preferred embodiments, treatment with the transforming agent resultsin the production of an "immortalized" hybridoma by means of fusing thesuspended spleen cells with mouse myeloma cells from a suitable cellline, e.g., SP-2, by the use of a suitable fusion promoter. Thepreferred ratio is about five spleen cells per myeloma cell in asuspension containing about 10⁸ splenocytes. A preferred fusion promoteris polyethylene glycol having an average molecule weight from about 1000to about 4000 (commercially available as PEG 1000, etc.); however, otherfusion promoters known in the art maybe employed.

The cell line is preferably of the so-called "drug resistant" type, sothat unfused myeloma cells will not survive in a selective medium, whilehybrids will survive. The most common class is 8-azaguanine resistantcell lines, which lack the enzyme hypoxanthine-guanine phosphoribosyltransferase and hence will not be supported by HAT (hypoxanthine,aminopterin, and thymidine) medium. It is also generally preferred thatthe myeloma cell line used be of the so-called "non-secreting" typewhich does not itself produce any antibody. In certain cases, however,secreting myeloma lines may be preferred.

(d) The transformed cells are then cloned, preferably to monoclonality.The cloning is preferably performed in a tissue culture medium that willnot sustain (support) non-transformed cells. When the transformed cellsare hybridomas, this is typically performed by diluting and culturing inseparate containers the mixture of unfused spleen cells, unfused myelomacells, and fused cells (hybridomas) in a selective medium which will notsustain the unfused myeloma cells. The cells are cultured in this mediumfor a time sufficient to allow death of the unfused cells (about oneweek). The dilution can be a limiting dilution, in which the volume ofdiluent is statistically calculated to isolate a certain number of cells(e.g., 1-4) in each separate container (e.g., each well of a microtiterplate). The medium is one (e.g., HAT medium) that will not sustain thedrug-resistant (e.g., 8-azaguanine resistant) unfused myeloma cell line.

(e) The tissue culture medium of the cloned transformants is analyzed(immunologically assayed) to detect the presence of antibody moleculesthat preferentially react with Fn and with a Fn polypeptide of thisinvention. This is accomplished using well known immunologicaltechniques.

(f) A desired transformant is then selected and grown in an appropriatetissue culture medium for a suitable length of time, followed byrecovery (harvesting) of the desired antibody from the culturesupernatant by well known techniques. The suitable medium and suitablelength of culturing time are also well known or are readily determined.

To produce a much greater concentration of slightly less pure monoclonalantibody, the desired hybridoma can be transferred by injection intomice, preferably syngeneic or semisyngeneic mice. The hybridoma willcause formation of antibody-producing tumors after a suitable incubationtime, which will result in a high concentration of the desired antibody(about 5-20 mg/ml) in the bloodstream and peritoneal exudate (ascites)of the host mouse.

Media and animals useful for the preparation of these compositions areboth well known in the art and commercially available and includesynthetic culture media, inbred mice and the like. An exemplarysynthetic medium is Dulbecco's minimal essential medium DMEM; Dulbeccoet al., Virol., 8:396 (1959)!supplemented with 4.5 gm/l glucose, 20 mMglutamine, and 20% fetal calf serum. An exemplary inbred mouse strain isthe Balb/c.

The monoclonal antibodies produced by the above method can be used, forexample, in diagnostic and therapeutic modalities wherein formation of aFn-containing immunoreaction product is desired. Methods for producinghybridomas that generate (secrete) antibody molecules having a desiredimmunospecificity, i.e., having the ability to immunoreact with aparticular protein, an identifiable epitope on a particular proteinand/or a polypeptide, but not immunoreact with a second polypeptide,such as the Eighth Type III repeat, are well known in the art and aredescribed further herein. Particularly applicable is the hybridomatechnology described by Niman et al., Proc. Natl. Acad. Sci. USA,80:4949-4953 (1983), and by Galfre et al., Meth. Enzymol., 73:3-46(1981), which descriptions are incorporated herein by reference.

A further preferred method for forming the instant antibody compositionsinvolves the generation of libraries of Fab molecules using the methodof Huse et al., Science, 246:1275 (1989). In this method, mRNA moleculesfor heavy and light antibody chains are isolated from the immunizedanimal. The mRNAs are amplified using polymerase chain reaction (PCR)techniques. The nucleic acids are then randomly cloned into lambdaphages to generate a library of recombined phage particles. The phagescan then be used to infect an expression host such as E. coli. The E.coli colonies and corresponding phage recombinants can then be screenedfor those producing the desired Fab fragments.

The antibody molecule-containing compositions employed in the presentinvention can take the form of solutions or suspensions. The preparationof a composition that contains antibody molecules as active ingredientsis well understood in the art. Typically, such compositions are preparedas liquid solutions or suspensions, however, solid forms suitable forsolution in, or suspension in, liquid can also be prepared. Thepreparation can also be emulsified. The active therapeutic ingredient isoften mixed with excipients which do not interfere with the assay andare compatible with the active ingredient. Suitable excipients are, forexample, water, saline, dextrose, glycerol, ethanol, or the like andcombinations thereof. In addition, if desired, the composition cancontain minor amounts of auxiliary substances such as wetting oremulsifying agents, pH buffering agents, and the like, which enhance theeffectiveness of the active ingredient.

An antibody molecule composition can be formulated into a neutralizedacceptable salt form. Acceptable salts include the acid addition saltsthat are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, 2-ethylamino ethanol,histidine, procaine, and the like.

EXAMPLES

The following examples are presented only for purposes of illustrationand in no way limit the invention.

1. Isolation of GPIIb-IIIa

The following procedure allows large scale preparation of plateletmembrane proteins from lysed platelets.

    ______________________________________                                        Reagents:               Final concentration                                   ______________________________________                                        Modified Tyrode's Buffer:                                                     50     ml     10x Tyrode's buffer 1x                                          0.5    g      crystalline BSA     1    mg/ml                                                (Sigma cat. #A-4378)                                            0.5    g      dextrose (d-glucose)                                                                              1    mg/ml                                  0.58   g      HEPES               10   mM                                     0.5    ml     1 M CaCl.sub.2      1    mM                                     2.5    ml     200 mM MgCl.sub.2   1    mM                                     bring to 500 ml with H.sub.2 O, and pH to 6.5 with HCl                        10X Tyrode's Buffer:                                                          160    g      NaCl                1.5  M                                      20.3   g      NaHCO.sub.3                                                     3.9    g      KCl                                                             Dissolve in 2 liters of double distilled H.sub.2 O                            Lysis Buffer:                                                                 0.348  g      HEPES               10   mM                                     4.8    g      NaCl                0.15 mM                                     4.42   g      Beta-octylglucoside 50   mM                                     0.3    ml     1 M CaCl.sub.2      1    mM                                     2      ml     200 mM MgCl.sub.2   1    mM                                     2      ml     150 mM PMSF in EtOH 1    mM                                     60     ul     50 mM leupeptin     10   mM                                     0.3    g      NEM (N-ethylmaleimide)                                                                            1    mg/ml                                  Bring to 300 ml volume with H.sub.2 O. Adjust pH to 7.4.                      ______________________________________                                    

Platelet packs from a blood bank are transferred to disposable 50 mlconical tubes (Falcon), balance and spun in Sorvall RT-6000 at 800 rpm(×g) for 10 minutes at 22° C. to pellet RBCs. Supernatants aretransferred to clean 50 ml conical tubes and spun at 2300 rpm for 20minutes at 22° C. The supernatant is transferred to autoclavable bottlefor disposal. Using a plastic 25 ml pipet and Modified Tyrode's Buffer,the platelet pellets are resuspended in one-half of the original volumeand spun again at 2300 rpm for 20 minutes at 22° C. The pellets areresuspended in Modified Tyrode's Buffer as before and spun again at 2300rpm for 20 minutes at 22° C. The platelet pellet is resuspended in Lysisbuffer (2 ml/platelet pack), spun in ultracentrifuge (SW 41 centrifugerotor) for 20 minutes at 20,000 rpm at 4° C., and the supernatant iscollected and stored frozen. Caution: The platelets should not beallowed to come in contact with glass and should always be kept at roomtemperature to avoid aggregation.

GPIIb-IIIa from platelet lysates is purified by the following procedure:

    ______________________________________                                        Reagents                                                                      RGD-Affinity chromatography column Buffer:                                    ______________________________________                                        0.348   g       HEPES            10   mM                                      4.8     g       NaCl             0.15 M                                       2.2     g       Beta-octylglucoside                                                                            25   mM                                      0.3     ml      1 M CaCl.sub.2   1    mM                                      1.5     ml      200 mM MgCl.sub.2                                                                              1    mM                                      2       ml      150 mM PMSF in EtOH                                                                            1    mM                                      60      ul      50 mM leupeptin  10   uM                                      0.3     g       NEM (N-ethylmaleimide)                                                                         1    mg/ml                                   Bring volume to just under 300 ml with H.sub.2 O, pH to 7.4,                  then adjust volume to 300 ml.                                                 Or use HEPES/NaCl Pre-made                                                    ______________________________________                                    

F7F-Sepharose Columns:

F7F-Sepharose: KYGRGDS-Sepharose (SEQ ID NO 15), 10 mg KYGRGDS/mlCNBr-activated Sepharose. 1 ml of F7F-Sepharose is loaded per column in7 ml disposable columns from Evergreen Scientific.

The following chromatographic procedure was employed: 8.1 ml columns ofF7F-Sepharose was equilibrated with 10 mls Lysis Buffer per column. Thecolumns were allowed to drain until surface of F7F-Sepharose is justexposed, then 1 ml of Platelet Lysate (1 ml Platelet Lysate-1 plateletpack) was applied.

The column was drained until the yellow color of the Platelet Lysatejust reaches the bottom of the column, cap both ends of the column andinvert overnight at 4° C. and the flowthrough was collected. The columnswere washed with 10 mls each of Column Buffer and the washes werecollected. The column was eluted with 2 mls per column of 1 mg/ml GRGDSP(SEQ ID NO 12) in Column Buffer followed by 2 mls of Column Buffer. Oneml fractions were collected and pooled. The concentration of purifiedGPIIb-IIIa collected was determined by its absorbance at 280 nm.

2. Preparation of Monoclonal Antibody Compositions

Monoclonal antibodies that immunoreact with a receptor binding site onfibronectin were produced using standard hybridoma technology withexceptions as noted. Briefly, two Balb/c mice were each immunizedintraperitoneally four times at one week intervals with increasing doses(1 mg, 10 mg, 25 mg, 50 mg and 100 mg, respectively) of immunogenconsisting of a mixture comprised of affinity-isolated GPIIb-IIIa, asprepared in Example 1 (1.25 mg/ml) and Fibronectin at 3 mg/ml. Theimmunogen was diluted 1:1 in Complete Freund's Adjuvant for the firstimmunization, in Incomplete Freund's Adjuvant for the second and thirdimmunization, and in normal saline for the fourth. Three days after thefourth immunization about 1×10⁸ lymphocytes were isolated from thespleens of both mice, admixed into a suspension and fused with 5×10⁷P3X63AG8.053 mouse myeloma cells using 50% PEG 1500 as the cell fusionpromoter. The resulting transformed (fused) antibody-producing cells(hybridomas) were initially transferred to 96-well microtiter plates ata density of about 1×10⁶ cells per well and cultured in selective HATmedia.

Tissue culture supernatants from about 2000 wells appearing to containviable HAT resistant hybridoma cells after 8 days of culturing werescreened in the ELISA assay for the presence of antibody molecules thatimmunoreact with fibronectin. The isolated hybridomas were thensubcloned twice at limiting dilutions to provide about 1 cell per welland 24 of the resultant hybridoma cultures were shown to be ofmonoclonal origin on the basis of two criteria: (1) each supernatant wasfrom a single cell foci and immunoreacted with fibronectin in the ELISAscreen, (2) each supernatant contained a single isotype ofimmunoglobulin when analyzed using the Mouse Ig Screening and IsotypingKit according to the instructions provided by the manufacturer,Boehringer-Mannheim Biochemicals, Indianapolis, Ind. The positivesupernatants were screened for their ability to inhibit binding of ¹²⁵I-fibronectin to GPIIb-IIIa coated microtiter cells. Positivesupernatants were screened in this way at each subcloning of hybridomas.The monoclonal antibody molecules were prepared by isolating theantibody molecules from the ascites fluid of a mouse using proteinA-Sepharose typically obtained from Pharmacia Inc. (Piscataway, N.J.)and used according to manufacturer's instructions. The proteinconcentration of isolated antibody molecule compositions as needed wasdetermined using the Bio-Rad Protein Assay Kit (Bio-Rad, Richmond,Calif.) according to the manufacturer's instructions.

To prepare a monoclonal antibody composition containing ¹²⁵ I-labeledantibody molecules, 350 microliters (μl) of PBS (0.15M NaCl, 0.01Msodium phosphate, pH 7.09) containing 1 milligram per milliliter (mg/ml)of the above isolated antibody molecules were admixed with 40 micrograms(μg) of chloramine-T and 1 millicurie (mCi) of carrier-free Na¹²⁵ I(Amersham, Arlington Heights, Ill.). The resulting admixture wasmaintained for 5 minutes at about 20° C. and then admixed with 20 μl ofa 2 mg/ml sodium metabisulfite solution (2 mg/ml) and 20 ul of apotassium iodide solution. Thereafter, 800 μl of PBS containing 1% BSAwere admixed followed by further admixture of diisopropylfluorophosphateto a final concentration of 10 mM. The resulting admixture wasmaintained for 60 minutes at 22° C. and then dialyzed against PBS. Thespecific activity of the resulting ¹²⁵ I-labeled antibody molecules wasabout 4.5 microcurie (uCi) per ug.

Compositions containing Fab fragments from the above isolated antibodymolecules were prepared by digestion with papain (200:1 weight perweight of Ig to papain) for 6 hours at 37° C. following the methods ofMage et al., Methods in Enzymolocy, 70:142-150 (1980). Undigested Ig andFc fragments were removed by chromatography on protein A-Sepharose. Theresulting Fab fragments-containing compositions were then ready for use,or were ¹²⁵ I-labeled, as needed, using the same procedures as describedabove for monoclonal antibody compositions.

3. ELISA Assays

a. ELISA To Screen Monoclonal Antibodies

Antibody molecules contained in hybridoma culture supernatants wereexamined for their ability to immunoreact with fibronectin. Fiftymicroliters (μl) of coating solution (0.1M NaHCO₃, pH 8.0, 0.1% NaN₃)containing 10 mg/ml of isolated fibronectin were admixed into the wellsof flat-bottom 96-well microtiter plates (Immulon 2; DynatechLaboratories, Chantilly, Va.). The plates were then maintained for 60minutes at 37° C. to permit the fibronectin to adsorb onto the walls ofthe wells. The coating solution was removed by shaking, the wells wererinsed twice with washing buffer (10 mM Tris-HCl at pH 7.4, 0.05% (v/v)TWEEN-20, 0.15M NaCl, and 200 mg/ml merthiolate), and 200 μl of blockingsolution (5% bovine serum albumin (BSA;w/v) in coating solution) wereadmixed into each well (solid support) to block excess protein sites.

The wells were maintained for 60 minutes at about 37° C. and then theblocking solution was removed. About 50 μl of hybridoma culturesupernatant diluted 1:1 in dilution buffer consisting of 0.1% (w/v) BSAin washing buffer was added to each well to form an immunoreactionadmixture. The resulting solid/liquid phase immunoreaction admixtureswere maintained at room temperature for 60 minutes to permit formationof a first solid phase-bound fibronectin-ligand complex and admixedantibodies. The solid and liquid phases were then separated, the wellswere rinsed twice with washing buffer, and excess liquid was removed byshaking.

Fifty μl of a solution containing horseradish peroxidase labeled goatanti-mouse IgG (Tago Inc., Burlingame, Calif.) diluted 1:1000 indilution buffer was admixed into each well to form a second solid liquidphase immunoreaction admixture (labeling immunoreaction admixture). Thewells were maintained for 60 minutes at room temperature to permitformation of a second immunoreaction product between the labeledantibody and any solid phase-bound antibody of the first immunoreactionproduct and then rinsed twice with washing buffer to isolate the solidphase-bound label-containing immunoreaction products. Excess liquid wasthen removed from the wells.

Fifty μl of freshly prepared chromogenic substrate solution containing4.0 mg/ml O-phenylenediamine and 0.012% (v/v) hydrogen peroxide in CPbuffer (243 μl of 0.1 m citric acid and 250 μl of 0.2M dibasic sodiumphosphate per liter H₂ O, pH 5.0) were then admixed into each well toform a color developing-reaction admixture. After maintaining the colordeveloping-reaction admixture for 10 minutes at about 20° C., 50 μl of2N H₂ SO₄ were admixed into each well to stop the developing-reaction,and the resulting solutions were assayed for absorbance at 490nanometers (nm) light wavelength using a Model 310 ELISA plate reader(Bio-Tek Instruments, Winooski, Vt.).

Antibody molecule compositions were considered to containanti-fibronectin immunoreactive antibody molecules if the measuredabsorbance at 490 nm (A490) was at least 6 times above background i.e.,above about 0.3 optical density units when measured at A490.

4. Screening Mab Inhibition of Fn-GPIIb-IIIa Interaction

The following microtiter well assay for binding of ¹²⁵ I-labeledfibronectin to GPIIb-IIIa was used:

Removable microtiter wells were coated with RGDS-affinity purifiedGPIIb-IIIa (50 μl at >10 mg/ml) at 4° C. The wells were blocked bydumping out the GPIIb-IIIa and incubating the wells in 150 μl 5% BSA for1-2 hrs. at room temperature. 25 μl of 25 nM ¹²⁵ I-labeled fibronectin(final concentration in well 12.5 nM) in 2× Modified Tyrodes waspre-incubated with 25 μl of hybridoma culture supernatant diluted 1:1 in10 mM Tris-HCl (pH 8) for 30 minutes at 37° C. in a separate microtiterwelltray. The GPIIb-IIIa coated and blocked microtiter wells were washed4 times with Modified Tyrodes Buffer. The ¹²⁵ I-fibronectin supernatantsolution was transferred to the GPIIb-IIIa coated wells and incubated atroom temperature for 4 hrs. The wells were washed 4 times with 200 μlModified Tyrodes. The empty wells were counted in a gamma counter andcpm/well determined.

5. Preparation and Isolation of Fn Fragment-MBP Fusion Proteins

Two MBP encoding plasmids, pPR734 and pIH 821, were employed as vectorsfor expressing the instant GPIIIa-MBP fusion proteins in E. coli. TheMBP region of the fused protein allows ready purification of the fusedproduct from other cellular proteins. The vectors were constructed viawell-known techniques following the procedures described hereinbelow.

A. Construction of MBP Vector

A cDNA clone containing the complete sequence of fibronectin (Fn) isdescribed by Obara et al, Cell, 53:649 (1988), and was provided by Dr.Yamada, an author on the publication. The provided cDNA clone wassubjected to restriction endonuclease digestion with PvuII, and theresulting digested fragments were blunt-end ligated with EcoRI linkersto adapt the digested fragments to contain EcoRI termini. The adaptedfragments were then digested with PstI to cleave those fragmentssusceptible to PstI (thereby inactivating the PstI-cleaved fragments forligation into an EcoRI site). The maltose binding fusion protein vectorpIH821 (New England Biolabs, Inc., Beverly, Mass.) and the adaptedfragments were both digested with EcoRI to produce EcoRI cohesivetermini, and the Fn fragment was ligated into the vector using DNA T4ligase to form a fusion construct having the cloned Fn-coding genefragment operatively linked to MBP-coding gene fragment capable ofexpressing a Fn-MBP fusion protein. The PvuII fragment contains theregion of Fn corresponding to the polypeptides P8-P9, namely, residues1255-1456. This construct is designated MBP/III8+9.

Vectors pIH821 and pPR734 are depicted in FIG. 5, and were obtained fromNew England Biolabs (Beverly, Mass.). The vectors each have a malElinked via a polylinker to a lac Z gene. pIH821 is identical to pPR734except that PIH821 has a deletion of the malE signal sequence 2-26,which facilitates export of fusion protein to the periplasm. The vectorseach have a tac promoter (Ptac) upstream of the malE gene. A lac I^(Q)suppressor gene immediately upstream of the tac promoter allowssuppression of expression activity until IPTG (isopropylβ-D-thiogalactoside) is used to induce expression. The remaining vectorbackbone is from Aval (filled in) to-Eco RI (filled in) of pKK233-2(Pharmacia, Piscataway, N.J.).

The important components of the maltose-binding protein fusionexpression system (MBP expression system) are the promoter (PtacII)previously described by Amann et al., Gene, 25:167-178 (1983); themaltose binding protein-laczα and fusion gene (malE-LacZα) previouslydescribed by Guan et al., Gene, 67:21-30 (1987); the rrn B ribosomaltranscription terminator previously described by Brosius et al., Proc.Natl. Acad. Sci. USA, 81:6929-6933 (1984) and commercially available inthe pIH821 and pPR734 vectors (New England Biolabs, Beverly, Mass.) andin the pKK223-3 and pKK233-2 (Pharmacia, Piscataway, N.J.). The MBPexpression system optionally contains the gene coding for the lacrepressor gene (lac I) previously described by Farabaugh, Nature,274:765-769 (1978). If the lac I gene is not present on the expressionvector it may be provided in trans by using the bacterial strainsexpressing the lambda repressor such as JM101, JM105, JM107, JM109 (ATCC#33323) and JM110 (ATCC #47013) described by Yanisch-Perron et al.,Gene, 33:103 (1985) which are commercially available from Stratagene (LaJolla, Calif.).

The individual nucleic acid segments containing the components of thisexpression system are operatively linked together (ligated) usingstandard molecular biology techniques, such as those described inMolecular Cloning: A Laboratory Manual, Second Edition, Sambrook et al.,eds, Cold Spring Harbor Laboratories, N.Y. (1989). When necessary, thereading frame of the various components is adjusted using syntheticlinkers, various fill-in reactions or various exonucleoses. In addition,various deletions and adjustments in the reading frame are easily madeusing loop-out mutagenesis and the commercially available mutagenesiskits such as the mutagene kit from Bio Rad Laboratories (Richmond,Calif.).

Each of the required components of the expression system will now bedescribed in detail. The Ptac II promoter previously described by Amannet al., Gene, 25:167-178 (1983) may be isolated from a number ofavailable vectors, such as PKK 223-3 (Pharmacia), PIH821 and pPR734 (NewEngland BioLabs, Beverly, Mass.), Ptac II (ATCC # 37245) and Ptac 12(ATCC #37138) described by Amann et al., Gene, 25:167-178 (1983). Forexample, the Ptac II promoter may be isolated using restrictionendonucleases from Ptac II using Eco RI and Hind III or ClaI and HindIII.

The maltose binding protein-lacZα fusion gene (malE-lacZα) previouslydescribed by Guan et al., Gene, 67:21-30 (1987) contains the malE geneon a Hinf I restriction endonuclease fragment isolated from thechromosome of an E. coli such as HB101 (ATCC # 33694) or wild type E.coli K12. The malE gene has been sequenced by Duplay et al., J. Biol.Chem., 259:10606-10613 (1984) and therefore probes specific for the malEgene may be easily synthesized allowing the malE gene to be isolatedfrom E. coli using standard cloning protocols. Alternatively, the malEgene may be chemically synthesized in segments and these segments joinedusing T4 DNA Ligase to produce the malE gene. The mature maltose bindingprotein (the malE gene product) is coded for by codons 28-342 of themalE gene sequence as described by Duplay et al., J. Biol. Chem.,259:10606-10613 (1984). The expression vector may either contain theentire malE gene coding for the 27 amino acid maltose binding proteinleader sequence and codons 28 to 392 coding for the mature maltosebinding protein or only the portion of the malE gene coding for themature maltose binding protein.

The remainder of the maltose binding protein-lacZα fusion gene containsthe portion of the lacZ gene coding for the shorter alpha (a) peptide ofthe lac gene (approximately 107 amino acids in length). This lacZ genemay be isolated from pUC 19 described by Yanisch-Perron et al., Gene,33:103-119 (1985) and is commercially available. The lacZ gene and themalE gene are linked together using a linker that may optionally havevarious useful restriction endonuclease recognition sequences in it.

The rrn B ribosomal transcription terminator previously described byBrosius et al., Proc. Natl. Acad. Sci. USA, 81:6929 (1984) and Brosiuset al., Plasmid, 6:112-118 (1981). The rrn B ribosomal transcriptionterminators may be easily isolated from available vectors, such asPEA300 (ATCC # 37181), PKK 223-3 and PKK 233-2 (Pharmacia), and PIH821and PPR734 (New England Biolabs). For example, the rrn B ribosomaltranscription terminator may be isolated from pKK223-3 using Hind IIIand Pvu I restriction endonucleases.

The lacI gene coding for the lambda repressor protein has been sequencedby Farabaugh, Nature, 274:765 (1978). In addition, the laci gene is inseveral available vectors, such as pBluescript II KS and pBluescript SK(Stratagene); and pUC 18 and pUC 19 (Pharmacia). The laci gene may beisolated from these vectors using restriction endonucleases and standardmolecular biology techniques. The laci gene may be present in theexpression vector or present in the bacteria the expression vector isgrown in.

The multiple cloning site present in the expression vector between themalE and lacZ genes is shown in Table 1 and listed in the SequenceListing as SEQ ID NO 14.

                  TABLE 1                                                         ______________________________________                                        Sequence of Polylinker in Expression System                                   Sac I Kpn I Eag I BamH I                                                      malE TCG AGC TCG GTA CCC GGC CGG GGA TCC ATC GAG                              Stu I Eco RI                                                                  GGT AGG CCT GAA TTC AGT AAA ACC CTC GAT                                       Factor X cleavage site                                                        BamH I Xba I Sal I Pst I Hind III                                             GGA TCC TCT AGA GTC GAC CTG CAG GCA AGC TTG lacZα                       Deletion of the malE Signal Sequence (FIG. 5):                                malE start codon                                                              ATG(D2-26)AAA ATC malE ...                                                    deletion of codons 2-26(signal sequence)                                      ______________________________________                                    

The multiple cloning site (polylinker) contains a nucleic acid segmentthat codes for a factor Xa cleavage site located between the malE andlacZ genes. Any maltose binding protein fusion protein produced by thisvector may be cleaved at the factor Xa cleavage site therebyfacilitating the purification of the desired protein.

Foreign genes may be inserted (operatively linked) into the multiplecloning sequence at the Sac I, Kpn I, Eag I or Bam HI restrictionendonuclease sites.

B. Expression of Fusion Protein

A small scale experiment is described to determine the behavior of aparticular MBP fusion protein. This protocol results in three crudeextract fractions; a total cell crude extract, a suspension of theinsoluble material from the crude extract, and a periplasmic fractionprepared by the cold osmotic shock procedure. Inoculate 80 ml richbroth+glucose & amp (per liter, 10 g Tryptone, 5 g yeast extract, 5 gNaCl, 1 g glucose, autoclave, add ampicillin to 100 μg/l) with cellscontaining the fusion plasmid produced above designated MBP/III 8+9.Grow at 37° C. with good aeration to 2×10⁸ cells/ml. Take a sample of 1ml and centrifuge for two minutes in a microfuge (uninduced cells).Discard supernatant and resuspend the cells in 100 μl SDS-PAGE samplebuffer. Vortex and place on ice.

Add IPTG (isopropylthiogalactoside) to the remaining culture to give afinal concentration of 0.3 mM, e.g., 0.24 ml of a 0.1M stock in H₂ O.Continue incubation at 37° C. for 2 hours. Take a 1 ml sample andcentrifuge for 2 minutes in a microfuge (induce cells). Discardsupernatant and resuspend the cells in 150 μl SDS-PAGE sample buffer.Vortex to resuspend cells and place on ice. (Additional time points at 1and 3 hours can be helpful in trying to decide when to harvest the cellsfor a large scale prep.)

Divide the culture into two aliquots and harvest the cells bycentrifugation at 4000×g for 10 minutes. Discard the supernatant andresuspend one pellet (sample A) in 5 ml 10 mM sodium phosphate, 30 mMNaCl, 0.25% Tween 20, 10 mM EDTA, 10 mM EGTA (Sigma E 4378), pH 7.0.Resuspend the other pellet (sample B) in 10 ml 30 mM Tris-HCl, 20%sucrose, pH 8.0 (9 ml for each 0.1 g cells wet weight). (The buffersspecified contain sodium phosphate, pH 7.0. The original buffers used inthe MBP affinity purification used Tris-HCl pH 7.2. However, Tris is avery poor buffer at pH 7.2, making it difficult to get predictablebuffers by dilution of concentrated stock solutions. Both buffers giveexcellent results, and other neutral buffers probably would work aswell).

Freeze Sample A in a dry ice-ethanol bath (or overnight at 20° C.) thenthaw in cold water. Sonicate the sample and monitor cell breakage bymeasuring the release of protein using the Bradford assay or A₂₈₀, untilit reaches a maximum. Centrifuge at 9,000×G for 20 minutes. Decant thesupernatant (crude extract 1) and save on ice. Resuspend the pellet in 5ml 10 mM sodium phosphate, 0.25% Tween 20, 30 mM NaCl, 10 mM EDTA, 10 mMEGTA, pH 7.0. This is a suspension of the insoluble matter (crudeextract 2). The reason for preparing three different extracts in thispilot experiment is to see the fusion 1) forms insoluble inclusionbodies, or 2) is exported to the periplasmic space. If the fusionprotein in insoluble, the protocol must be modified to produce solubleprotein (see below). If the fusion is efficiently exported, preparationof a periplasmic fraction should be considered as an alternative topreparing a crude cellular extract. Preparation of a periplasmic extractgives a substantial purification by itself.

Add EDTA to 1 mM of Sample B and incubate for 5-10 minutes at roomtemperature with shaking or stirring. Centrifuge at 4° C., remove allthe supernatant, and resuspend the pellet in 10 ml ice-cold 5 mM MgSO₄.Shake or stir for 10 minutes in an ice bath. Centrifuge at 4° C. Thesupernatant is the cold osmotic shock fluid.

Add 5 μl 2× SDS-PAGE sample buffer to 5 μl of crude extracts 1 & 2 and10 μl 2× SDS-PAGE sample buffer to 10 μl of the cold osmotic shockfluid. Boil these samples, along with the uninduced and induced cellsamples, for 5 minutes. Centrifuge in a microfuge for 2 minutes. Load 20μl of the uninduced and induced cells samples, and all of the extractsamples, on a 10% SDS-PAGE gel. Optionally, one can run an identicalSDS-PAGE gel(s) using 1:5 dilutions of the above samples, prepare aWestern blot and develop with anti-MBP serum and, if available, serumimmunospecific for the cloned portion of the fusion protein. Anotherpilot to optimize export of the fusion protein, i.e. find the besttemperature (23°, 30°, or 37° C.) and IPTG level, and the best time toharvest the cells, may be desirable. If the fusion protein is ininsoluble matter, make sure that the cells are completely broken. If itis still insoluble, try extracting the pellet with 0.2% Triton X-100, 1mM EDTA a few times; often the protein is not truly insoluble but justassociated with the membrane fragments in the cell pellet. If this isthe case, be aware that, for some fusions, Triton interferes withbinding to the column. If the protein is truly insoluble, modify theconditions of cell growth to attempt to produce soluble fusion protein.Three changes that have helped in previous cases are i) changing to adifferent strain background, ii) growing the cells at 23° C. or 30° C.,and iii) inducing with 0.01 mM IPTG to give lower expression levels(Takagi et al., Biotechnology, 6:948 (1988)).

C. Purification of Fusion Protein

(1) Preparation of Cross-linked Amylose Resin

For 300 ml resin, place 10 g. amylose (Sigma, Catalog No. A-7043), 40 mlH₂ O and a stirring bar in a 1000 ml beaker and warm to 50° C. withstirring. In a fume hood, add 60 ml 5N NaOH, then 30 ml epichlorhydrin(Sigma, Catalog No. E-4255) with rapid stirring. The suspension willheat up upon gelling. Continue stirring until the suspension forms asolid gel, about 10 minutes (it should turn a little yellow). Let coolto room temperature (about 45 minutes--1 hour) then cut the gel intopieces and wash three times with 1000 ml H₂ O. Transfer the gel to aWaring blender and fragment the gel for about 3-5 s. Wash with 1000 ml50 mM glycine-HCl, 0.5M NaCl, pH 2.0, two times and discharge the finesbetween washes.

Wash with water 3 times (keep discharging fines), then with 10 mM sodiumphosphate , pH 7.0, three times. Suspend the gel in 10 mM sodiumphosphate, 0.02% sodium azide, pH 7.0 and store at 4° C. Blocknon-specific sites on the resin by washing in 1000 ml 3% non-fat drymilk overnight at 4° C.

(2) Affinity Chromatoaraphy

The following are protocols for large scale purification of a hybridfusion protein.

Inoculate 1 liter rich broth, glucose and ampicillin (per liter, 10 gTryptone, 5 g yeast extract, 5 g NaCl, 1 g glucose, autoclave, addampicillin to 100 μg/ml) with cells containing the fusion plasmid. Growto 2×10⁸ cells/ml (A₆₀₀ of 0.4). Add IPTG to a final concentration of0.3 mM, e.g. 85 mg or 3 ml of a 0.1M stock in H₂ O. Incubate the cellsat 37° C. for 1-3 hours. (The period of time to allow for expressiondepends on the host used and whether the hybrid protein is unstable, andshould be determined empirically.) Harvest the cells by centrifugationat 4000×g and resuspend in 50 ml 10 mM sodium phosphate, 30 mM NaCl,0.2% Tween 20, 10 mM β-mercaptoethanol, 10 mM EDTA, 10 mM EDTA, 10 mMEGTA (Sigma, Catalog Number E 4378), pH 7.0. Freeze the sample in a dryice-ethanol bath (or overnight at 20° C.) and thaw in cold water.Sonicate and monitor cell breakage, by measuring the release of proteinusing the Bradford assay or A₂₈₀, until it reaches a maximum. Centrifugeat 9,000×g for 30 minutes. (For many unstable proteins, most of thedegradation happens during harvest and cell breakage. Therefore, it isbest to do it quickly and keep the cells chilled. Fifty ml of lysisbuffer is based on the expectation of about 5 grams cells/liter, i.e.,10 ml for every gram of cells (wet weight)).

The EGTA is to help inhibit proteases that have a Ca++ cofactor.Addition of PMSF (phenyl methyl-sulfonylfluoride) and other proteaseinhibitors can be tried on a case to case basis. β-mercaptoethanol isincluded to prevent interchain disulfide bond formation upon lysis(disulfide bonds usually do not form intracellularly in E. coli) if theprotein sensitive to EGTA, 0.5M NaCl or mercaptoethanol, adjust thebuffer accordingly.

Pour the cross-linked amylose resin into an Erlenmeyer flask and let itsettle. Wash the resin in at least an equal volume of columnbuffer+0.25% Tween-20 a few times; column buffer=10 mM sodium phosphate,0.5M NaCl, pH 7.0 (optional: 10 mM β-mercaptoethanol, 1 mM EGTA). Pour acolumn of about 40-200 ml resin for each liter of culture and wash thecolumn with 3 column volumes the same buffer. (The amount of resindepends on the resin and the amount of hybrid protein produced."Homemade" resin binds at about 0.5 to 1 mg/ml bed volume, so for ayield of 40 mg/l you need an 80 ml column. Because the flow propertiesof the homemade resin are poor, a short fat column works best. Columnshape is less important for this resin since it is in the form of beads;column height to diameter ratios of 4 perform well.)

Dilute the crude extract 1:5 with column buffer+0.25% Tween-20. Load thediluted crude extract at a flow rate of 10×(diameter of column in cm)²!ml/hr. This is about 1 ml/min for a 2.5 cm column. The dilution of thecrude extract is aimed at reducing the protein concentration to about2.5 mg/ml. A good rule of thumb is that 1 g wet weight of cells givesabout 120 mg protein.

The crude extract can be passed through the column twice to be sure thatall the MBP hybrid is bound to the column, but in most cases all thefusion that is competent to bind does so on the first pass. Fusionprotein can also be loaded on the resin batch-wise, by incubating crudeextract and resin at 4° C. for 2-76 h with gentle agitation. Wash with 3column volumes column buffer +0.25% Tween-20 then wash with 5 columnvolumes column buffer without Tween-20. Elute the hybrid protein with 10mM sodium phosphate, 0.5M NaCl, 10 mM maltose, pH 7.0 (optional: 10 mMβ-mercaptoethanol, 1 mM EGTA). Collect 10-20 fractions each=to 1/5th to1/10th the column volume and assay the fractions for protein, e.g., bythe Bradford assay or A₂₈₀ ; the fractions containing the MBP hybridshould have easily detectable protein. The hybrid protein elutesdirectly after the void volume of the column. Pool theprotein-containing fractions. (Optional) Dialyze vs. 4×100 volumes 10 mMTris-Cl, 100 mM NaCl, (optional: 1 mM EGTA) pH 8.0 to remove maltose.Concentrate in an Amicon Centricon or Centriprep concentrator, an Amiconstirred-cell concentrator, or the equivalent.

If the MBP domain is separated from the target peptide by cleavage withFX₈ and amylose affinity chromatography, dialyze to get rid of themaltose in your hybrid protein. In this situation, the rate at whichligand dialyzes away is inversely proportional to the concentration ofthe binding protein (Silhavy et al, 1975). Therefore, it is best todialyze at a fusion protein concentration of 200 μg/ml or less, and thenconcentrate the fusion afterwards.

6. Inhibition of Fn/Fg-GPIIb-IIIa Integration by Fusion Protein

96-well Immulon-2 microtiter plates (Dynatech-Immulon) were coated with50 μl of RGD-affinity purified GPIIb-IIIa, diluted to 10 μg/ml in 10 mMHepes, 0.15M NaCl, 1 mM CaCl₂ 1 mM MgCl₂.

GPIIb-IIIa:

RGD-Affinity purified GPIIb-IIIa in Column Buffer with GRGDSP:

10 mM HEPES

0.15M NaCl

1 mM CaCl₂

1 mM MgCl₂

1 mg/ml N-ethylmaleimide

10 uM leupeptin

1 mM PMSF (phenyl methylsulfonyl fluoride)

25 mM Beta-Octylglucoside

1 mg/ml GRGDSP (SEQ ID NO 12)

Incubate the GPIIb-IIIa with plate for 1 hour at room temperature, thendump unbound and block with 150 ul of 5% BSA in the same buffer for 1hour at room temperature. Wash three times with 150 μl of Tyrode'sbuffer with 5 mM HEPES, 1 mM MgCl₂ 1 mg/ml dextrose, 1 mg/ml BSA. Add 50μl of ¹²⁵ I fusion protein (125 mg/ml) at varied concentration (4×10⁻⁷to 1×10⁻¹⁰ M). Incubate 4 hours to overnight at room temperature (withlead shield).

Carefully remove radioactive samples and wash 3 times with 150 μlTyrodes with 5 mM HEPES, 1 mM MgCl₂, 1 mg/ml dextrose, 1 mg/ml BSA.Count with a Gamma Counter and elute by boiling in SDS-PAGE loadingBuffer.

In an alternative embodiment, non-radioactive Fg may be used instead ofhot, and detected by reacting with an appropriate anti-Fg antibody andHRP-conjugated secondary antibody. Using this variation, each antibodyis incubated with the washed plate for 1 hr. The anti-Fg antibody willhave to be titrated, but a 1:1000 dilution of HRP-conjugated antibodyfrom most manufacturers works fine. The advantage of using radioactiveFg is that the number of bound Fg molecules may determined based on thespecific activity. If the amount of GPIIb-IIIa that adheres to theplastic is quantitated as well, then the stoichiometry of the bindingcan be determined.

7. Regeneration of Fn Polypeptides from Fusion Protein

Factor Xa cleavage of fusions is carried out at a w/w ratio of 1 or 2%the amount of fusion protein. Depending on the particular fusionprotein, ratios of 0.1% to 5% will work as well. The reaction mixturecan be incubated for 3 hours to 1 day, at room temperature or 4° C.Again depending on the particular fusion, it may be necessary todenature the fusion to render the factor Xa site accessible to cleavage.This can be accomplished by dialyzing in (or adding) guanidinehydrochloride to 6M, then dialyzing against the factor Xa cleavagebuffer.

If necessary, dialyze the fusion protein is dialyzed against 20 mMTris-HCl, 100 mM NaCl, pH 8.0 (=factor Xa cleavage buffer) and a pilotexperiment is performed with a small portion of the protein. Forexample, 20 μl fusion protein at 1 mg/ml is mixed with 1 μl factor Xa at200 μg/ml.

In a separate tube, place 5 μl fusion protein with no factor Xa.Incubate the tubes at room temperature. At 2, 4, 8, and 16 H, take 5 μlof the factor Xa reaction, add 5 μl 2× SDS-PAGE sample buffer and saveon ice. Prepare a sample of 5μl fusion protein=5 μl 2× sample buffer.Boil the samples for 5 minutes and run on an SDS-PAGE gel.

The pilot experiment can be scaled up exactly for the portion of thefusion protein to be cleaved. A small sample of the uncut fusion issaved as a reference and complete cleavage by SDS-PAGE is checked.

To denature the fusion protein, dialyze the fusion against 20 mMTris-HCl, 6M guanidine hydrochloride, pH 8.0. Dialyze against 20 mMTris-HCl, 100 mM NaCl, pH 8.0. Stepwise dialysis against this buffercontaining decreasing amounts of guanidine hydrochloride can preventprecipitation of the fusion protein. Halving the guanidine concentrationat each step is convenient; cases where 0.1M steps are necessary havebeen reported.

8. Discussion of Examples 1-7

In order to identify the sites of fibronectin that participate ininteraction with the platelet integrin, GPIIb-IIIa, monoclonalanti-fibronectin antibodies were raised and screened for their abilityto inhibit fibronectin binding to highly purified GPIIb-IIIa. Threeantibodies able to inhibit such interaction are depicted in FIG. 4Aalong with a control monoclonal (Mab 15). In this figure the dose ofantibody added is presented as the abscissa, and the percent binding offibronectin to purified GPIIb-IIIa (fibronectin present at 50 nM) is onthe ordinate. FIG. 4B shows a cross competition experiment in which theradiolabelled antibodies are indicated above and the "cold" competingantibody is indicated below. The results indicate that FnI-8 recognizesan epitope distinct from the other antibodies and that FnI-11 and 16cross-compete with each other and therefore recognize the same epitope.

                  TABLE 2                                                         ______________________________________                                        Mab Interactions with Fibronectin                                             Polypeptides Encoded by cDNA Fragments                                        Base      Fibronectin.sup.+                                                                           Mab*                                                  Seq. ID No.                                                                             Residue Sequence                                                                            8     11    12   16                                   ______________________________________                                        3          934-1653     +     +     +    +                                    3         1317-1653     +     +     +    +                                    3         1359-1653     +     +     +    +                                    3         1163-1399     -     -     -    -                                    3         1410-1653     +     +     ND   +                                    3         1255-1456     +     +     +    +                                    3         1351-1456     +     +     ND   +                                    3         1378-1456     -     -     ND   -                                    ______________________________________                                         *ND =  Not Determined; 8 = FnI8; 11 = FnI11; 12 = FnI12; 16 = FnI16 (IgG      Kappa)                                                                        .sup.+ Kornblihtt et al. EMBO J 4:1755 (1985)                            

The epitopes of these monoclonal antibodies were mapped by expressingvarious fragments of fibronectin in bacteria utilizing the prokaryoticexpression vector lambda GT11. Table 2 presents the fibronectin type IIIrepeats encoded by the CDNA. All monoclonals reacted with a whole cDNAexpressed protein in which the RGDS sequence in the tenth type IIIrepeat had been deleted (not shown). They also reacted with a fragmentcontaining residues 1255-1456 which does not contain RGDS (SEQ ID NO12). All inhibitory antibodies also reacted with a deletion containingresidues 1351-1456. These data indicate that certain monoclonalantibodies against fibronectin, which inhibit its binding to GPIIb-IIIa,react with a fragment whose carboxy terminus begins at least 50 aminoacids upstream of the RGDS sequence.

To determine the region of fibronectin that contains the epitopes thatinteract with GPIIb-IIIa and inhibit fibronectin binding, theconstruction containing bases encoding the Fn fragment 1255-1456 wasexpressed as a fusion protein with a maltose binding protein in aplasmid vector. This fusion protein was readily purified on acrosslinked amylose column, and the capacity of this fusion protein toinhibit fibronectin binding to purified GPIIb-IIIa was assessed.

In FIG. 6, a fusion protein containing fibronectin residues 1255-1456(open circles) inhibited fibronectin binding to purified GPIIb-IIIa. Ona weight basis, the material was about four fold less potent than intactfibronectin, but it has numerous contaminants in addition to the insertcoded polypeptide. In comparison, BSA, or the maltose binding proteinalone, lacked inhibitory activity.

To further assess the nature of the inhibitor material, the mixture offusion protein and maltose binding protein breakdown products was passedthrough a monoclonal FnI-16 immunoaffinity column and eluted at lowpH+6M urea. The starting, pass through, and bound and eluted fractionswere analyzed for the capacity to inhibit fibronectin binding toGPIIb-IIIa, and analyzed by SDS-PAGE Coommassie blue staining, andwestern blotted with monoclonal antibody FnI-16. Passage of the fusionprotein mixture over the anti-fibronectin monoclonal antibody columnresulted in quantitative removal of inhibitory activity which could bepartially recovered in the low pH+Urea eluate. In contrast, passagethrough an irrelevant monoclonal antibody had no such effect. Inspectionof the stained gels shows that the starting material was depleted of thetwo higher molecular weight bands by passage through theanti-fibronectin affinity column indicating that the bands are reactivewith the monoclonal antibody.

To determine if the insert coded polypeptide containing fibronectinresidues 1255-1456 bound GPIIb-IIIa, the capacity of microtiter wellscoated with purified GPIIb-IIIa to bind the radiolabelled fusion-proteinpreparation was ascertained (Table 3). In addition, the radiolabelledmaterial bound to the insolubilized GPIIb-IIIa was recovered andanalyzed by SDS-PAGE followed by radioautography. The radiolabelled Fnfusion protein bound to insolubilized GPIIb-IIIa and the binding wasspecific and inhibited by monoclonal antibodies FnI-16 and EDTA. Incontrast, there was no such binding when radiolabelled maltose bindingprotein alone was employed. Specific binding of fibronectin, inhibitableby the monoclonal antibody (Mab 16.12) and EDTA, was also observed(Table 3). The isolated protein products were a mixture of insert codedpolypeptides containing fusion protein and maltose binding proteinbreakdown products. only those bands containing the insert-codedpolypeptide bound to the insolubilized GPIIb-IIIa and that binding wasinhibitable by either EDTA or the monoclonal antibody FnI-16. Inaddition, no binding was observed to BSA coated wells. In contrast, themaltose binding protein alone failed to specifically bind to theGPIIb-IIIa coated wells.

                  TABLE 3                                                         ______________________________________                                        Interaction of .sup.125 I-Labelled                                            Ligands for GPIIb-IIIa                                                        Ligand*       Inhibitor  Counts ×10.sup.-3                              ______________________________________                                        MBP/8-9       --         124                                                                Mab 16.12  18                                                                 2 mM EDTA   4                                                   MBP           --         15                                                                 Mab 16.12   9                                                                 2 mM EDTA  -4                                                   Fibronectin   --         42                                                                 Mab 16.12  12                                                                 2 mM GDTA   8                                                   ______________________________________                                         *MBP = Maltose binding protein                                                MBP/89 = Maltose binding protein/8-9 fusion protein                      

The capacity of the fusion protein to inhibit fibrinogen binding toGPIIb-IIIa also wa s assessed. The fusion protein was observed to be anefficient inhibitor of fibrinogen binding to GPIIb-IIIa, whereas themaltose binding protein was not. The fusion protein also was observed toinhibit fibrinogen (Fg) binding to GPIIb-IIIa (FIG. 7).

In sum, these data directly indicate that the insert-coded polypeptidehas the capacity to bind to GPIIb-IIIa specifically, to inhibit thebinding of fibrinogen and fibronectin, and thus is predicted to be aninhibitor of cell adhesive events, such as platelet aggregation. To testthis hypothesis, the interaction of cells with fibronectin was examinedin the presence of the fusion protein.

Thus, the fact that the insert-coded polypeptide binds directly toGPIIb-IIIa indicates that it alone, or in conjunction with an RGDsequence, could be used to promote cell attachment in clinicalsituations such as wound healing, prosthesis implantation, or seeding ofendothelial grafts.

9. Characterization of Novel Peptide Inhibitors of Fibronectin andFibrinogen Binding to Integrin Receptor GPIIb-IIIa

As shown in Example 8, the 202 amino acid residue fragment infibronectin (Fn) beginning at residue 1255 and extending to residue 1456contained the integrin receptor GPIIb-IIIa binding site. The fragmentcompetitively inhibited the binding to both fibronectin and fibrinogento the receptor as shown in Example 8. To determine the minimum receptorbinding region within the 202 amino acid residue sequence, syntheticpolypeptides and variants thereof were generated and evaluated for theirability to bind to the receptor and competitively inhibit the binding oflabelled ligand to the receptor. The approaches used in thecharacterization of the minimum receptor binding site are describedbelow.

A. Preparation of Synthetic Polypeptides

Synthetic polypeptides of various lengths spanning the region of humanFn beginning at amino acid residue 1255 and ending with 1456 wereprepared at the Scripps Clinic and Research Foundation Peptide SynthesisCore Facility (La Jolla, Calif.) using the classical solid-phasetechnique described by Merrifield, Adv. Enzymol., 32:221-296 (1969) asadapted for use with a model 430 automated peptide synthesizer (AppliedBiosystems, Foster City, Calif.). Prepared polypeptide resins werecleaved by hydrogen fluoride, extracted and analyzed for purity byhigh-performance liquid chromatography (HPLC) using a reverse-phase C18column manufactured by Waters Associates, Milford, Mass.

B. Characterization of a GPIIb-IIIa-Specific Receptor BindingPolypeptide

(1) Inhibition of Fn Binding to Purified GPIIb-IIIa by a Fn-DerivedPolypeptide

Polypeptides synthesized in Example 9a were evaluated for their abilityto competitively inhibit the binding to Fn to purified GPIIb-IIIaintegrin receptor. The competition assays were performed as described byCharo et al., J. Biol. Chem., 266:1415-1421 (1991). The competitionassay was performed by first admixing into individual wells of a 96 wellmicrotiter plate (Immunlon, Dynatech) 10 μg/ml of purified GPIIb-IIIa,prepared in Example 1, diluted in HEPES-saline buffer, pH 7.4,consisting of 10 mM HEPES, 150 mM NaCl, 1 mM CaCl₂ and 1 mM MgCl₂. Theplates were maintained for 16 hours at 4° C. to allow the purifiedreceptor to adsorb onto the walls of the wells. The receptor-coatedwells were then washed two times with Modified Tyrode's buffer, preparedas described in Example 1, to remove any non-bound receptor from thereceptor-coated wells. Non-receptor-occupied sites on the microtiterwells were blocked by admixing 5% BSA dissolved in Modified Tyrode'sbuffer into each well and maintaining the plate for 2 hours at roomtemperature.

After removing the blocking solution and washing the blocked wells asdescribed above, 50 μl of a 10 nM solution of biotinylated Fn inModified Tyrode's buffer was admixed into each well in the presence ofpolypeptides prepared in Example 9a ranging in concentration from 0.1 μMto 200 μM polypeptide to form a solid-liquid phase receptor-proteinadmixture. Fn admixed in the absence of polypeptides served as apositive control for the competition assay. Purified Fn was prepared asdescribed by Plow et al., J. Biol. Chem., 256:9477-9482 (1981) and U.S.Pat. No. 4,589,981.

Briefly, Fn was isolated from fresh human citrated plasma by affinitychromatography on gelatin-sepharose as described by Engvall et al., Int.J. Cancer, 20:1-5 (1977). Bound Fn was eluted with 1M sodium borate atpH 5.3 and the major eluted protein peak was dialyzed against 0.15MNaCl, 0.01M sodium phosphate of pH 7.0. The isolated Fn yielded a singleband on SDS-PAGE under nonreducing conditions and a closely spaceddoublet of 215,000 to 230,000 molecular weight under reducingconditions.

The resultant purified Fn was then biotinylated as described by Dale etal., Blood, 77:1096-1099 (1991). Briefly, for biotinylation, Fn wasfirst dialyzed into 0.1M NaHCO₃ containing 0.1M NaCl at pH 8.0 andcentrifuged at 100,000×g for 30 minutes at 4° C. to remove anyparticulate matter. The protein concentration was adjusted to 0.5 mg/mlin 50 mM sodium borate at pH 8.5. The biotinylation reaction wasinitiated by admixture of N-hydroxysuccinimido biotin (NHS-biotin; 0.5mg/mg protein) (Pierce Biochemicals, Rockford, Ill.) followed bymaintenance of the admixture for 2 hours at room temperature to formbiotinylated Fn. The resultant biotinylated Fn was then dialyzed againstTris-HCl-saline to remove remaining salts.

The Fn-peptide admixtures were maintained in GPIIb-IIIa-coated wells for2 hours at room temperature to allow the biotinylated Fn to bind to thereceptor. Following this maintenance period, the reacted wells werewashed as described above and the amount of biotinylated Fn bound toGPIIb-IIIa was determined by admixing 0.1 ml avidin bound tobiotinylated horseradish peroxidase H (Hrp) (Sigma, St. Louis, Mo.) at a1:2000 dilution to form an avidin-biotinylated Fn admixture. Theadmixture was maintained for 60 minutes at room temperature to allowformation of an avidin Hrp-biotinylated Fn complex. Excess avidin Hrpwas removed by washing as described and the presence of biotinylated Fnbound to purified GPIIb-IIIa was detected by admixture of 50 μl offreshly prepared chromogenic substrate solution containing 4.0 mg/mlO-phenylenediamine and 0.012% (v/v) hydrogen peroxide in CP buffer asdescribed in Example 3a. After maintaining the color developing-reactionadmixture for 10 minutes at 20° C., the reaction was stopped with theadmixture of 2N H₂ SO₄, and the stopped reactions were measured forabsorbance as described in Example 3a.

The amount of biotinylated Fn bound to purified GPIIb-IIIa in theabsence of competitor polypeptides measured at an absorbance of 490 nmyielded an absorbance of 2.70. The polypeptide designated D-11-T havingthe amino acid residue sequence DRVPHSRNSIT, SEQ ID NO 11, whichcorresponds to the Fn amino acid residue sequence beginning at residuenumber 1394 and ending at residue number 1404, competitively inhibitedthe binding of Fn to purified GPIIb-IIIa as shown in FIG. 8. Maximalinhibition was achieved with approximately 200 μM polypeptideconcentration. The inhibition curve paralleled the inhibition of Fnbinding to receptor by RGDS polypeptide which effected maximalinhibition at 10 μM concentration. Thus, the synthetic polypeptideD-11-T derived from a non-RGD containing region of Fn competitivelyinhibited the binding of Fn to GPIIb-IIIa.

A polypeptide according to the formula of SEQ ID NO 16 was also preparedas in Example 9a and shown in the above assay to inhibit Fn binding toGPIIb-IIIa by at about 35-40% of control binding when tested at 200 μMpolypeptide. Thus, the polypeptide DRVPHSR (SEQ ID NO 16) defines thenon-RGD site on Fn of this invention that inhibits for binding toGPIIb-IIIa.

(2) Inhibition of Fn Binding to Purified GPIIb-IIIa by Variants ofDRVPHSRNSIT Polypeptide

To determine the specificity with which the polypeptide D-11-T(DRVPHSRNSIT) (SEQ ID NO 11) inhibited the binding of Fn to GPIIb-IIIa,competition assays were performed as described in Example 9b(1) in thepresence of a scrambled polypeptide VHPDRNTISRS (SEQ ID NO 13) or with11 separate polypeptides containing an alanine substitution at one ofthe 11 amino acid residues at positions from 1394 to 1404. The scrambledand 11 variant polypeptides were prepared as described in Example 9a foruse in this assay.

The assays were performed as described in Example 9b(1) except that thesynthetic polypeptides were used at a concentration of 250 μM. Theresults of the competition assays with either scrambled oralanine-substituted variant polypeptides as compared with the inhibitionobtain by the D-11-T polypeptide are shown in FIG. 9. The results shownin the bar graph represent the average plus/minus the standard deviationof three determinations. In contrast with the D-11-T polypeptide whichinhibited the binding of biotinylated Fn to GPIIb-IIIa by greater than80%, the scrambled polypeptide was ineffective at inhibiting Fn bindingto the receptor. In addition, the variant polypeptides substituted withalanine for the aspartic acid amino acid residue at position 1394 andfor the arginine amino acid residue at position 1395 were both equallyineffective at inhibiting the binding to Fn to GPIIb-IIIa.

Several other polypeptides containing alanine substitutions were alsowithout effect. Specifically, alanine substituted for arginine atresidue position 1400 had the next least inhibitory effect followed bythe histidine at residue number 1398, then by proline at residue number1397; by valine at residue number 1396 and lastly by serine at residuenumber 1399. Alanine substitutions for either the asparagine (1401),serine (1402), isoleucine (1403) or threonine (1404) did not result in adecrease of inhibitory effectiveness of the polypeptide in preventingthe binding of Fn to GPIIb-IIIa. Thus, alanine substitutions near thecarboxy-terminal portion of the D-11-T polypeptide did not alter theinhibitory activity of the polypeptide whereas substitutions near theamino-terminal end altered the inhibitory activity.

(3) Inhibition of Fn or Fa Binding to Purified GPIIb-IIIa or AlDhav/Beta 3 by DRVPHSRNSIT Polypeptide

To determine the receptor specificity of the D-11-T polypeptide,competition assays were performed as described in Example 9b(1) usingeither biotinylated fibronectin (Fn) or fibrinogen (Fg) and eitherGPIIb-IIIa or alphav/beta3 (αVβ₃). The latter is another integrinreceptor, also designated as the vitronectin receptor, which hasRGD-dependent ligand binding sites for both Fn and Fg. See Charo et al.,J. Cell Biol., 111:2795-2800 (1990). These assays were performed tosupport the findings above that the D-11-T polypeptide inhibits thebinding of ligands to their receptors in an RGD-independent manner.

The αVβ₃ receptor used in this assay was isolated as described by Smithet al., J. Biol. Chem., 263:18726-18731 (1988). Briefly, vitronectinreceptor was purified from human placenta, the diced tissue of which wasfirst extracted with 100 mM octylglucoside, 2 mM CaCl₂, 1 mMphenylmethylsulfonyl fluoride in PBS for 30 minutes at room temperature.The extract was filtered and then pumped over a monoclonal antibody, LM609, coupled to Affi-gel (Bio-Rad, Richmond, Calif.). Bound vitronectinreceptor was eluted from the column with 0.01M acetic acid at pH 3.0containing 0.1% Nonidet P-40 and 2 mM CaCl₂. Fractions collected thatcontained vitronectin receptor were pooled and dialyzed against PBS.Vitronectin receptor purified by this method was greater than 90% pureas judged by Coomassie Blue Staining of SDS-PAGE and typical yields were500-900 μg of receptor/placenta.

Human Fg was purified by the glycine precipitation procedure describedby Kazal et al., Proc. Soc. Exp. Biol. Med., 113:989-994 (1963) fromfresh-frozen plasma that was treated with 10 units/ml heparinimmediately after thawing. Following the final glycine precipitation, Fgwas dialyzed against 50 mM Tris-HCl, 100 mM NaCl, 0.02% NaN₃ at pH 7.4and stored at -80° C. until used. Purified Fg was biotinylated asdescribed for Fn in Example 9b(1). Fn and GPIIb-IIIa were prepared asdescribed in Examples 9b(1) and 1, respectively. The assay was performedessentially as described in Example 9b(1) with the exception that 5 nmof biotinylated Fg and 10 nM of biotinylated Fn were used. The assayswere performed on microtiter wells coated with either GPIIb-IIIa or withαVβ₃ in the presence of polypeptide D-11-T or the scrambled polypeptideVHPDRNTISRS (SEQ ID NO 13).

The results of the competition assays described above are shown in FIG.10 in two panels, 10A and 10B. FIG. 10A and 10B shows, respectively,that the polypeptide D-11-T specifically inhibited the binding of Fg andFn to GPIIb-IIIa-coated wells as indicated by the line with opensquares. The scrambled polypeptide, indicated by the line shown withclosed squares, failed to exhibit an inhibitory effect on the binding ofeither Fg or Fn to GPIIb-IIIa. In αVβ₃ -coated wells, the D-11-Tpolypeptide as well as the scrambled polypeptide failed to exhibit anyinhibitory activity as shown in FIG. 10A with the lines indicated byopen and closed circles, respectively. This lack of inhibition was alsofound in αVβ₃ -coated wells with Fn as shown in FIG. 10B.

These results confirm that the polypeptide D-11-T is specific for theGPIIb-IIIa integrin receptor inhibiting the binding of both Fn and Fg tothe receptor. The advantage that this polypeptide has as compared toexisting RGD polypeptides is that it is GPIIb-IIIa-specific asdetermined by the lack of inhibitory effect of preventing Fn and Fg frombinding to the vitronectin receptor, αVβ₃. Thus, the specific advantagesof the D-11-T polypeptide amino acid residue sequence as a model ofnovel antithrombotics is, 1) it is a novel GPIIb-IIIa inhibitorypolypeptide and 2) it is specific for GPIIb-IIIa versus αVβ₃.

By making modifications including substitutions or cyclization, theactivity of the D-11-T polypeptide sequence may be enhanced. Inaddition, this amino acid residue sequence or its derivatives may haveother properties which may make it a more desirable antithrombotic thanexisting RGD polypeptides.

Although the present invention has been described in some detail by wayof illustration and example for purposes of clarity and understanding,it will be obvious that certain modifications may be practiced withinthe scope of the appended claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 22                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: internal                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: 3                                                               (D) OTHER INFORMATION: /note= "xaa can be any amino acid                      residue"                                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /note= "xaa can be any amino acid                      residue"                                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       AspArgXaaProHisXaaArg                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: internal                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: 3                                                               (D) OTHER INFORMATION: /note= "xaa is an amino acid that                      can be either V or A."                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /note= "xaa is an amino acid that                      can be either S or A."                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: 8                                                               (D) OTHER INFORMATION: /note= "xaa is an amino acid that                      can be either N or A."                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "xaa is an amino acid that                      can be either S or A."                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: 10                                                              (D) OTHER INFORMATION: /note= "xaa is an amino acid that                      can be either I or A."                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: 11                                                              (D) OTHER INFORMATION: /note= "xaa is an amino acid that                      can be either T or A."                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       AspArgXaaProHisXaaArgXaaXaaXaaXaa                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: internal                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: 3                                                               (D) OTHER INFORMATION: /note= "xaa is an amino acid that                      can be either V or A."                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /note= "xaa is an amino acid that                      can be either S or A."                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: 8                                                               (D) OTHER INFORMATION: /note= "xaa is an amino acid that                      can be either N or A."                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       AspArgXaaProHisXaaArgXaaSerIleThr                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: internal                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: 3                                                               (D) OTHER INFORMATION: /note= "xaa is an amino acid that                      can be either V or A."                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /note= "xaa can be an amino acid                       that can be either S or A."                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "xaa is an amino acid that                      can be either S or A."                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       AspArgXaaProHisXaaArgAsnXaaIleThr                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: internal                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: 3                                                               (D) OTHER INFORMATION: /note= "xaa is an amino acid that                      can be either V or A."                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /note= "xaa is an amino acid that                      can be either S or A."                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: 10                                                              (D) OTHER INFORMATION: /note= "xaa is an amino acid that                      can be either I or A."                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       AspArgXaaProHisXaaArgAsnSerXaaThr                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: internal                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: 3                                                               (D) OTHER INFORMATION: /note= "xaa is an amino acid that                      can be either V or A."                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /note= "xaa is an amino acid that                      can be either S or A."                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: 11                                                              (D) OTHER INFORMATION: /note= "xaa is an amino acid that                      can be either T or A."                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       AspArgXaaProHisXaaArgAsnSerIleXaa                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 106 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       ProThrGlyIleAspPheSerAspIleThrAlaAsnSerPheThrVal                              151015                                                                        HisTrpIleAlaProArgAlaThrIleThrGlyTyrArgIleArgHis                              202530                                                                        HisProGluHisPheSerGlyArgProArgGluAspArgValProHis                              354045                                                                        SerArgAsnSerIleThrLeuThrAsnLeuThrProGlyThrGluTyr                              505560                                                                        ValValSerIleValAlaLeuAsnGlyArgGluGluSerProLeuLeu                              65707580                                                                      IleGlyGlnGlnSerThrValSerAspValProArgAspLeuGluVal                              859095                                                                        ValAlaAlaThrProThrSerLeuLeuIle                                                100105                                                                        (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 202 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       ProAlaValProProProThrAspLeuArgPheThrAsnIleGlyPro                              151015                                                                        AspThrMetArgValThrTrpAlaProProProSerIleAspLeuThr                              202530                                                                        AsnPheLeuValArgTyrSerProValLysAsnGluGluAspValAla                              354045                                                                        GluLeuSerIleSerProSerAspAsnAlaValValLeuThrAsnLeu                              505560                                                                        LeuProGlyThrGluTyrValValSerValSerSerValTyrGluGln                              65707580                                                                      HisGluSerThrProLeuArgGlyArgGlnLysThrGlyLeuAspSer                              859095                                                                        ProThrGlyIleAspPheSerAspIleThrAlaAsnSerPheThrVal                              100105110                                                                     HisTrpIleAlaProArgAlaThrIleThrGlyTyrArgIleArgHis                              115120125                                                                     HisProGluHisPheSerGlyArgProArgGluAspArgValProHis                              130135140                                                                     SerArgAsnSerIleThrLeuThrAsnLeuThrProGlyThrGluTyr                              145150155160                                                                  ValValSerIleValAlaLeuAsnGlyArgGluGluSerProLeuLeu                              165170175                                                                     IleGlyGlnGlnSerThrValSerAspValProArgAspLeuGluVal                              180185190                                                                     ValAlaAlaThrProThrSerLeuLeuIle                                                195200                                                                        (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 606 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..606                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       CCAGCTGTTCCTCCTCCCACTGACCTGCGATTCACCAACATTGGTCCA48                            ProAlaValProProProThrAspLeuArgPheThrAsnIleGlyPro                              151015                                                                        GACACCATGCGTGTCACCTGGGCTCCACCCCCATCCATTGATTTAACC96                            AspThrMetArgValThrTrpAlaProProProSerIleAspLeuThr                              202530                                                                        AACTTCCTGGTGCGTTACTCACCTGTGAAAAATGAGGAAGATGTTGCA144                           AsnPheLeuValArgTyrSerProValLysAsnGluGluAspValAla                              354045                                                                        GAGTTGTCAATTTCTCCTTCAGACAATGCAGTGGTCTTAACAAATCTC192                           GluLeuSerIleSerProSerAspAsnAlaValValLeuThrAsnLeu                              505560                                                                        CTGCCTGGTACAGAATATGTAGTGAGTGTCTCCAGTGTCTACGAACAA240                           LeuProGlyThrGluTyrValValSerValSerSerValTyrGluGln                              65707580                                                                      CATGAGAGCACACCTCTTAGAGGAAGACAGAAAACAGGTCTTGATTCC288                           HisGluSerThrProLeuArgGlyArgGlnLysThrGlyLeuAspSer                              859095                                                                        CCAACTGGCATTGACTTTTCTGATATTACTGCCAACTCTTTTACTGTG336                           ProThrGlyIleAspPheSerAspIleThrAlaAsnSerPheThrVal                              100105110                                                                     CACTGGATTGCTCCTCGAGCCACCATCACTGGCTACAGGATCCGCCAT384                           HisTrpIleAlaProArgAlaThrIleThrGlyTyrArgIleArgHis                              115120125                                                                     CATCCCGAGCACTTCAGTGGGAGACCTCGAGAAGATCGGGTGCCCCAC432                           HisProGluHisPheSerGlyArgProArgGluAspArgValProHis                              130135140                                                                     TCTCGGAATTCCATCACCCTCACCAACCTCACTCCAGGCACAGAGTAT480                           SerArgAsnSerIleThrLeuThrAsnLeuThrProGlyThrGluTyr                              145150155160                                                                  GTGGTCAGCATCGTTGCTCTTAATGGCAGAGAGGAAAGTCCCTTATTG528                           ValValSerIleValAlaLeuAsnGlyArgGluGluSerProLeuLeu                              165170175                                                                     ATTGGCCAACAATCAACAGTTTCTGATGTTCCCAGGGACCTGGAAGTT576                           IleGlyGlnGlnSerThrValSerAspValProArgAspLeuGluVal                              180185190                                                                     GTTGCTGCGACCCCCACCAGCCTACTGATC606                                             ValAlaAlaThrProThrSerLeuLeuIle                                                195200                                                                        (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 606 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      GATCAGTAGGCTGGTGGGGGTCGCAGCAACAACTTCCAGGTCCCTCGGAACATCAGAAAC60                TGTTGATTGTTGGCCAATCAATAAGGGACTTTCCTCTCTGCCATTAAGAGCAACGATGCT120               GACCACATACTCTGTGCCTGGAGTGAGGTTGGTGAGGGTGATGGAATTCCGAGAGTGGGG180               CACCCGATCTTCTCGAGGTCTCCCACTGAAGTGCTCGGGATGATGGCGGATCCTGTAGCC240               AGTGATGGTGGCTCGAGGAGCAATCCAGTGCACAGTAAAAGAGTTGGCAGTAATATCAGA300               AAAGTCAATGCCAGTTGGGGAATCAAGACCTGTTTTCTGTCTTCCTCTAAGAGGTGTGCT360               CTCATGTTGTTCGTAGACACTGGAGACACTCACTACATATTCTGTACCAGGCAGGAGATT420               TGTTAAGACCACTGCATTGTCTGAAGGAGAAATTGACAACTCTGCAACATCTTCCTCATT480               TTTCACAGGTGAGTAACGCACCAGGAAGTTGGTTAAATCAATGGATGGGGGTGGAGCCCA540               GGTGACACGCATGGTGTCTGGACCAATGTTGGTGAATCGCAGGTCAGTGGGAGGAGGAAC600               AGCTGG606                                                                     (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      AspArgValProHisSerArgAsnSerIleThr                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      GlyArgGlyAspSerPro                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      ValHisProAspArgAsnThrIleSerArgSer                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 96 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      TCGAGCTCGGTACCCGGCCGGGGATCCATCGAGGGTAGGCCTGAATTCAGTAAAACCCTC60                GATGGATCCTCTAGAGTCGACCTGCAGGCAAGCTTG96                                        (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      LysTyrGlyArgGlyAspSer                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      AspArgValProHisSerArg                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      AspArgValProHisSerArgAsnSerIleThr                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      AspArgValProHisAlaArgAsnSerIleThr                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      AspArgValProHisSerArgAlaSerIleThr                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      AspArgValProHisSerArgAsnAlaIleThr                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      AspArgValProHisSerArgAsnSerAlaThr                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      AspArgValProHisSerArgAsnSerIleAla                                             1510                                                                          __________________________________________________________________________

What is claimed is:
 1. An isolated polypeptide consisting of the aminoacid residue sequence AspArgXaa₁ ProHisXaa₂ Arg (SEQ ID NO:1), whereinXaa₁ and Xaa₂ are any amino acid residue and said polypeptide bindsGPIIb-IIa in an ArgGlyAsp-independent manner.
 2. The polypeptide ofclaim 1 wherein said polypeptide has the amino acid residue sequenceAspArgValProHisSerArg (SEQ ID NO 16).
 3. An isolated polypeptideconsisting of the amino acid residue sequence AspArgXaa₁ ProHisXaa₂ArgXaa₃ Xaa₄ Xaa₅ Xaa₆ (SEQ ID NO:2), wherein Xaa₁ is Val or Ala, Xaa₂is Ser or Ala, Xaa₃ is Asn or Ala, Xaa₄ is Ser or Ala, Xaa₅ is Ile orAla, and Xaa₆ is Thr or Ala.
 4. The polypeptide of claim 3 wherein saidpolypeptide has the amino acid residuesequenceAspArgValProHisSerArgAsnSerIleThr,AspArgAlaProHisSerArgAsnSerIleThr, AspArgValProHisAlaArgAsnSerIleThr,AspArgValProHisSerArgAlaSerIleThr, AspArgValProHisSerArgAsnAlaIleThr,AspArgValProHisSerArgAsnSerAlaThr, andAspArgValProHisSerArgAsnSerIleAla,the SEQ ID NO of which are 11, 17, 18,19, 20, 21 and 22, respectively.